DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

BACKGROUND: Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied in the diagnosis of bacteria. METHODS AND RESULTS: We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species: Escherichia coli, Candida albicans, Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. CONCLUSIONS: Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

Evaluation of conjunctival swab as a mass-screening tool for molecular diagnosis of canine visceral leishmaniasis.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil once the dog is the main reservoir host of the disease. The aim of this study was to evaluate the conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1350 domiciled dogs were screened. The animals were evaluated by serological tests (enzyme-linked immunosorbent assay (ELISA) as screening and immunofluorescence antibody test (IFAT) for confirmation) and by CS associated to real-time PCR, using primers addressed to kinetoplast DNA (kDNA) minicircles and SYBR Green. Canine ß-globin gene amplification was used to evaluate the sample DNA integrity. A subgroup of 484 animals was also submitted to clinical evaluation. Among the 1350 dogs screened, 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty-one percent (39/126) of the ELISA-positive dogs were confirmed by IFAT. CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL. The results demonstrated that CS real-time PCR was able to detect a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.

Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84.

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m ((99m)Tc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with (99m)Tc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA-), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with (99m)Tc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.

Foot-and-mouth disease virus: DNA aptamer selection for the 3ABC protein.

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis. Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

Gamma radiation effects on Sporothrix schenckii yeast cells.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

Evaluation of the specific uptake of radiolabeled Staphylococcus aureus aptamers in the infectious foci.

The specific uptake of 99mTc radiolabeled Staphylococcus aureus aptamers in the infectious foci was evaluated by scintigraphic imaging of infection-bearing mice. The radiotracer uptake was inhibited by non-radiolabeled aptamers in a competition assay. In addition, when a different number of bacterial cells was used to infect mice an increase in the target/non-target ratios of images correlated with the increase of CFU per gram of tissue was verified. These results confirmed that 99mTc-aptamers were specific to bacterial focus and the level of uptake was dependent on the number of bacterial cells.

Mice immunization with radioattenuated yeast cells of Paracoccidiodes brasiliensis: influence of the number of immunizations.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America. Up to the moment no vaccine has been reported. The aim of this study was to evaluate the influence of the number of immunizations on the protection elicited by radioattenuated yeast cells of P. brasiliensis. BALB/c mice were divided into two groups that were immunized once (Group 1) or twice (Group 2), respectively. In each group, mice were divided into sub-groups that were challenged 30, 45, or 60 days after the second immunization. Organ colony-forming units (CFUs) was determined 90 days post-challenge. A significant reduction in CFUs recovery was verified in both groups, but it was higher in Group 2. Histologic alterations were observed only in Group 1. The cytokines IL-4, IL-10, and IFN-gamma were produced in mice of Group 1. In Group 2, only IFN-gamma was significantly detected. IgG2a predominance relative to IgG1 was also observed in Group 2. Altogether, our results indicated that mice immunized once developed a mixed Th1/Th2 response, which was less efficient in the infection control, while a trend to a Th1 pattern was obtained with two immunizations, promoting optimal elimination of P. brasiliensis yeast cells from mice tissues.

Detection of mixed Leishmania infections in dogs from an endemic area in southeastern Brazil.

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.

Evaluation of the conjunctival swab for canine visceral leishmaniasis diagnosis by PCR-hybridization in Minas Gerais State, Brazil.

The visceral leishmaniasis (VL) in Brazil is caused by Leishmania chagasi (L. infantum) and dogs are considered to be the main domestic reservoir. The epidemiological control involves the elimination of infected dogs. Therefore, the correct diagnosis is very important in order to avoid the disease transmission or unnecessary culling of dogs. Recently, an antileishmanial vaccine for dogs was licensed and commercialized in Brazil. Vaccinated dogs test positive in the conventional serological tests, rendering these assays useless for control programs involving vaccinated animals. The Polymerase Chain Reaction (PCR) is an attractive alternative to the diagnosis in this context; but non-invasive samplings have great importance because they are simpler, painless and less resisted by dog-owners. This study aimed at evaluating the conjunctival swab (CS) for canine VL diagnosis by the PCR-hybridization procedure. Two groups of 23 seropositive dogs were used. CS samples were obtained from both eyes of each animal. The DNA extraction from CS was performed by the phenol chloroform method in group 1 and by boiling in group 2. In addition, blood was collected from each animal so that 30 microl was spotted onto filter paper (FP) and 1.0 ml was treated to obtain the buffy coat (BC). The DNA extraction from the BC and FP was accomplished by identical procedures in both groups using commercial kits. The PCR positivities for both groups 1 and 2 were, respectively: 73.9% and 52.2% (CS), 13% and 30.4% (BC), 8.7% and 17.4% (FP). The hybridization step increased the positivities for: 91.3% and 65.2% (CS), 21.7% and 34.8% (BC), 30.4% and 43.5% (FP), respectively. The highest frequency of positivity was obtained by the association between CS and DNA extraction by phenol chloroform. This approach can be very useful for diagnosis of canine leishmaniasis and could be applied to the follow-up and regular screening of vaccinated dogs.

Mouse Immunization with Radioattenuated Yeast Cells of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent mycosis in Latin America, and currently there is no effective vaccine. The present chapter describes the methodology to obtain radioattenuated yeast cells of P. brasiliensis and a protocol to evaluate protective response elicited by this immunogen in experimental paracoccidioidomycosis. The radioattenuated yeast provides a valuable tool for immunological studies in experimental paracoccidioidomycosis and vaccine research.

Detection of bacterial infection by a technetium-99m-labeled peptidoglycan aptamer.

Nuclear medicine clinicians are still waiting for the optimal scintigraphic imaging agents capable of distinguishing between infection and inflammation, and between fungal and bacterial infections. Aptamers have several properties that make them suitable for molecular imaging. In the present study, a peptidoglycan aptamer (Antibac1) was labeled with 99mTc and evaluated by biodistribution studies and scintigraphic imaging in infection-bearing mice. Labeling with 99mTc was performed by the direct method and the complex stability was evaluated in saline, plasma and in the molar excess of cysteine. The biodistribution and scintigraphic imaging studies with the 99mTc-Antibac1 were carried out in two different experimental infection models: Bacterial-infected mice (S. aureus) and fungal-infected mice (C. albicans). A 99mTc radiolabeled library, consisting of oligonucleotides with random sequences, was used as a control for both models. Radiolabeling yields were superior to 90% and 99mTc-Antibac1 was highly stable in presence of saline, plasma, and cysteine up to 6h. Scintigraphic images of S. aureus infected mice at 1.5 and 3.0h after 99mTc-Antibac1 injection showed target to non-target ratios of 4.7±0.9 and 4.6±0.1, respectively. These values were statistically higher than those achieved for the 99mTc-library at the same time frames (1.6±0.4 and 1.7±0.4, respectively). Noteworthy, 99mTc-Antibac1 and 99mTc-library showed similar low target to non-target ratios in the fungal-infected model: 2.0±0.3 and 2.0±0.6for 99mTc-Antibac1 and 2.1±0.3 and 1.9 ± 0.6 for 99mTc-library, at the same times. These findings suggest that the 99mTc-Antibac1 is a feasible imaging probe to identify a bacterial infection focus. In addition, this radiolabeled aptamer seems to be suitable in distinguishing between bacterial and fungal infection.

(1→3)-ß-D-glucan aptamers labeled with technetium-99m: Biodistribution and imaging in experimental models of bacterial and fungal infection.

INTRODUCTION: Acid nucleic aptamers are RNA or DNA oligonucleotides capable of binding to a target molecule with high affinity and selectivity. These molecules are promising tools in nuclear medicine. Many aptamers have been used as targeting molecule of radiopharmaceuticals in preclinical studies. (1→3)-ß-D-glucans are the main structural cell wall components of fungi and some bacteria. In the present study two radiolabeled (1→3)-ß-D-glucan aptamers (seq6 and seq30) were evaluated to identity infectious foci caused by fungal or bacterial cells. METHODS: Aptamer labeling with 99mTc was performed by the direct method and biodistribution studies were accomplished in Swiss mice (n=6) infected in the right thigh muscle with Staphylococcus aureus or Candida albicans. A 99mTc radiolabeled library consisting of oligonucleotides with random sequences was used as control. RESULTS: There was a higher uptake of 99mTc radiolabeled aptamers in the infected thigh than in the left thigh muscle (non-infected) in the S. aureus infected animals. The target/non-target ratios were 3.17±0.22 for seq6 and 2.66±0.10 for seq30. These ratios were statistically higher than the value (1.54±0.05) found for the radiolabeled library (control). With regard to biodistribution, no statistical difference was verified between aptamers and control uptakes in the infection foci in the C. albicans infected animals. The target/non-target ratios were 1.53±0.03, 1.64±0.12 and 1.08±0.02 for radiolabeled library, seq6 and seq30, respectively. Scintigraphic imaging of infected foci using radiolabeled aptamers was possible only for S. aureus infected mice. CONCLUSIONS: Seq6 and seq30 aptamers proved to be inefficient for diagnosis of C. albicans infection. Nevertheless, their applicability for diagnosis of S. aureus and other bacterial infections by scintigraphy should be further explored.

Scintigraphic imaging of Staphylococcus aureus infection using 99mTc radiolabeled aptamers.

Staphylococcus aureus is a specie of great medical importance associated with many infections as bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device related infections. Early identification of infectious foci is crucial for successful treatment. Scintigraphy could contribute to this purpose since specific radiotracers were available. Aptamers due to their high specificity have great potential for radiopharmaceuticals development. In the present study scintigraphic images of S. aureus infectious foci were obtained using specific S. aureus aptamers radiolabeled with 99mTc.

Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Brazil.

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.

Identification of Staphylococcus aureus infection by aptamers directly radiolabeled with technetium-99m.

INTRODUCTION: Aptamers are oligonucleotides that have high affinity and specificity for their molecular targets which are emerging as a new class of molecules for radiopharmaceuticals development. In this study, aptamers selected to Staphylococcus aureus were evaluated for bacterial infection identification. METHODS: Anti S. aureus aptamers were labeled with (99m)Tc by the direct method. The radiolabel yield and complex stability were assessed by thin-layer chromatography (TLC). Three groups of Swiss mice containing 6 animals each were used. The first group was infected intramuscularly in the right thigh with S. aureus. The second group was infected in the same way with C. albicans and the third group was injected with zymosan to induce aseptic inflammation. After 24 h, radiolabeled aptamers (22.2 MBq) were injected by the tail vein. The mice were euthanized 4 h post injection and tissue sample activities measured in a gamma counter. RESULTS: The (99m)Tc labeled aptamers were stable in saline, plasma and cystein excess. Radiolabeled aptamers showed increased uptake in the kidneys for all groups indicating a main renal excretion, which is consistent with the hydrophilic nature and small size of aptamers. The radiopharmaceutical showed rapid blood clearance indicated by a reduced dose (% ID/g) in the blood. The biodistribution showed that aptamers were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 4.0±0.5. This ratio for mice infected with C. albicans was 2.0±0.4 while for mice with aseptic inflammation was 1.2±0.2. Histology confirmed the presence of infection in groups 1 and 2, and inflammation in group 3. CONCLUSIONS: The biodistibution study demonstrated a statistically higher uptake in the S. aureus foci relative to inflammation and C. albicans infected areas. These results highlight the potential of aptamers labeled directly with (99m)Tc for bacterial infection diagnosis by scintigraphy.

Selection of peptidoglycan-specific aptamers for bacterial cells identification.

Peptidoglycan is a highly complex and essential macromolecule of bacterial outer cell wall; it is a heteropolymer made up of linear glycan strands cross-linked by peptides. Peptidoglycan has a particular composition which makes it a possible target for specific bacterial recognition. Aptamers are single-stranded DNA or RNA oligonucleotides that bind to target molecules with high affinity and specificity. Aptamers can be labeled with different radioisotopes and possess several properties that make them suitable for molecular imaging. The purpose of this study was to obtain aptamers for use as radiopharmaceutical in bacterial infection diagnosis. Two aptamers (Antibac1 and Antibac2) against peptidoglycan were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology. The dissociation constant (Kd) for Antibac1 was 0.415 + 0.047 µM and for Antibac2 was 1.261 + 0.280 µM. These aptamers labeled with (32)P showed high affinity for Staphylococcus aureus cells. The binding to S. aureus and Escherichia coli in vitro were significantly higher than for Candida albicans and human fibroblasts, demonstrating their specificity for bacterial cells. These results point Antibac1 and Antibac2 as promising tools for bacterial infections identification.

Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

Nasal, oral and ear swabs for canine visceral leishmaniasis diagnosis: new practical approaches for detection of Leishmania infantum DNA.

BACKGROUND: The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify Leishmania infantum DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P=0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05). CONCLUSIONS: Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil.

BACKGROUND: We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. METHODOLOGY/PRINCIPAL FINDINGS: Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. CONCLUSIONS: The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.

PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples.

The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.