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ABSTRACT: The objective of this study was to develop a novel liposomal formulation, containing phosphatidylserine (PS), of buparvaquone (BPQ) and to evaluate its in vivo effectiveness in Leishmania (L.) infantum chagasi-infected hamsters. The activity of BPQ was evaluated against both the promastigote forms of different Leishmania species and the intracellular amastigotes of L. (L.) infantum chagasi. Buparvaquone was entrapped in PS-liposomes (BPQ-PS-LP), and the drug was quantified by ultra-high-performance liquid chromatography. The treatment was quantified by detecting the RNA of the living amastigotes in the spleen and the liver by real-time PCR. In vitro assays with L. (L.) infantum chagasi intracellular amastigotes were performed in peritoneal macrophages for the evaluation of the 50% inhibitory concentration (IC(50)). BPQ-PS-LP at 0.33 mg/kg/day for eight consecutive days reduced the number of amastigotes by 89.4% (P<0.05) in the spleen and by 67.2% (P>0.05) in the liver, compared to 84.3% (P<0.05) and 99.7% (P<0.05), respectively, following Glucantime® treatment at 50 mg/kg/day. Free BPQ at 20 mg/kg/day failed to treat the hamsters when compared to the untreated group. BPQ was significantly (P<0.05) selective against L. (L.) infantum chagasi intracellular amastigotes, with an IC(50) value of 1.5 μM; no in vitro mammalian cytotoxicity could be detected. Other cutaneous species were also susceptible to BPQ, with IC(50) values in the range 1-4 μM. BPQ-PS-LP caused a significant reduction in the parasite burden at a 60-fold lower dose than did the free BPQ. These results show the potential of PS-liposome formulations for the successful targeted delivery of BPQ in visceral leishmaniasis.
Experimental Parasitology 03/2012; 130(3):195-9. · 2.12 Impact Factor
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ABSTRACT: Cerebral toxoplasmosis is the most common neurologic opportunistic infection in HIV-infected patients. Excretory-secretory antigens (ESA) are the majority of the circulating antigens in sera from hosts with acute toxoplasmosis, and their usefulness as antigens has been shown. This study considered whether it could find anti-ESA antibodies in cerebrospinal fluid (CSF) and whether these antibodies can be markers of active infection. Samples of CSF from 270 HIV-infected patients were analyzed and divided into 3 groups according to the presence or absence of active toxoplasmosis. Group I: 99 patients with cerebral toxoplasmosis; group II: 112 patients with other opportunistic neurologic diseases and seropositive for toxoplasmosis; and group III: 59 patients with other opportunistic neurologic diseases and seronegative for toxoplasmosis. Toxoplasma gondii ESA and a crude tachyzoite antigen were used as antigens using ELISA and immunoblotting. The statistical analysis was done using the F test and unpaired Student's t test. Crude tachyzoite antigen: mean ELISA-relative values ± standard error for CSF of groups I and II were 7.0 ± 0.27 and 3.9 ± 0.19, respectively. Variance analysis revealed that results of both groups of patients were statistically different (1.80, P = 0.0025). The difference between the mean results was 3.0 ± 0.3, and the Student's t test value was 9.41 (P = 0.0001). Samples from groups I and II were reactive by immunoblotting, with similar intensities. In ESA-ELISA, the mean for group I was 9.0 ± 0.39. Group II showed a mean value of 2.7 ± 0.12. Both groups were statistically different (9.16, P < 0.001). However, in ESA, the difference between the mean results was higher (6.2 ± 0.39) and the Student's t test value was 16.04 (P < 0.0001). Similar results were shown in immunoblotting where a CSF sample from group I reacted well with ESA, and the sample from a group II patient failed to do so. The mean ELISA-relative value of the control group (group III) was 0.5 ± 0.09 for the first antigen and 0.4 ± 0.22 for the second. ESA-ELISA and/or immunoblotting of CSF samples can be used for diagnosis of cerebral toxoplasmosis in association with clinical, serologic, and radiological information, thus providing a simple straightforward methodology, particularly suitable in countries with high prevalence of latent toxoplasmosis in the general population.
Diagnostic microbiology and infectious disease 09/2011; 71(3):279-85. · 2.45 Impact Factor
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ABSTRACT: This prospective study evaluated the value of laboratorial diagnosis in ocular toxoplasmosis analyzing peripheral blood samples from a group of Brazilian patients by immunologic and molecular methods. We analyzed blood samples from 184 immunocompetent patients with ocular disorders divided into 2 groups: Group I, composed of samples from 49 patients with ocular toxoplasmosis diagnosed by clinical features; Group II, samples from 135 patients with other ocular diseases. Samples were assayed by conventional polymerase chain reaction (cnPCR), real-time PCR (qPCR) for Toxoplasma gondii, indirect immunofluorescence reaction (IF), avidity test (crude tachyzoite lysate as antigen), and excreted-secreted tachyzoite proteins as antigen (ESA-ELISA). cnPCR and qPCR profiles were concordant in all samples. Positive PCR was shown in 40.8% of group I patients. The majority of the positive blood samples (75%) were taken from patients with toxoplasmic retinochoroiditis scars, and the others (25%), from patients with retinal exudative lesions. Despite that 86 of the 135 patients from Group II had asymptomatic toxoplasmosis, all DNA blood samples had negative PCR. Concordant results were shown in the data obtained by serologic methods. Around 24% of the patients with ocular toxoplasmosis had high antibody titers determined by ESA-ELISA and IF. Anti-ESA antibodies are shown principally in patients with active infection. Collectively, these data demonstrate the presence of tachyzoites in the blood of patients with chronic infection, supporting the idea of recurrent disease. Circulating parasites in blood of immunocompetent individuals may be associated with the reactivation of the ocular disease.
Diagnostic microbiology and infectious disease 07/2011; 70(3):362-6. · 2.45 Impact Factor
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ABSTRACT: The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC(50)) values of 3.81 and 21.55 μM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC(50) values in the range of 3 to 7 μM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC(50) value of 62.67 μM. However, bepridil showed lack of activity at 12 mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.
Experimental Parasitology 02/2011; 128(2):111-5. · 2.12 Impact Factor
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ABSTRACT: The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.
Parasitology Research 01/2011; 109(2):267-74. · 2.15 Impact Factor
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ABSTRACT: Toxoplasma gondii is an important food-borne parasite transmitted primarily from animals to humans through meat consumption, mainly pork and lamb, as well as through oocysts shed by cats. Infection in humans can cause severe neonatal malformations, ocular complications or encephalitis. Toxoplasmosis infection during pregnancy, especially in sheep, often results in abortion, representing considerable economic loss. The aim of this study was to investigate whether Toxoplasma gondii pooled excreted-secreted antigens (ESA), recovered from infected culture supernatants with tachyzoites used as immunogen, can protect experimental mice against T. gondii infection. For immunization experiments, we evaluated A/Sn inbred mice, a novel susceptible mouse model for T. gondii and a virulent strain (RH) for challenge experiments. The antigen selection was based on those produced by tachyzoites since they are responsible for disseminating the infection as well as stimulating the humoral and cellular immune responses. ESA were recovered from VERO cell-culture supernatants infected with virulent RH strain tachyzoites harvested after 48 h. Groups of 5 female mice were intraperitoneally (i.p.) immunized with 4 doses at 2 week intervals with 20 microg of ESA adsorbed to 0.5 mg of alum. The control group received only the adjuvant in PBS on the same dates. Pooled serum collected from chronically infected mice was used as positive control. Blood samples were collected from tail veins 14 days after each immunization. Antibody was detected using ELISA, indirect immunofluorescence and immunoblotting. Anti-ESA antibodies were also evaluated by agglutination, complement-mediated lysis and antibody-mediated cellular toxicity. Fifteen days after the last immunization, both groups were challenged (i.p.) with 1 x 10(3) RH strain tachyzoites. The parasitemia was evaluated by PCR, and survival was followed daily. The results showed an increase of antibody levels after each immunization. Anti-ESA antibodies also reacted with a crude tachyzoite antigen and bonded on the parasite surface, with particularly high intensity at the apical region. Anti-ESA antibodies were also able to agglutinate and kill tachyzoites in vitro through interactions with complement and cellular pathways. Even though the tachyzoite challenge was lethal to the mice, PCR results suggested that immunized mice had lower parasitemia as well as longer survival (72 h) than mice from the control group.
Experimental Parasitology 08/2008; 120(3):227-34. · 2.12 Impact Factor
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ABSTRACT: Despite the development of serological and molecular methods in recent years, the diagnosis of cerebral toxoplasmosis in human immunodeficiency virus (HIV)-infected patients still presents difficulties. In the present study, we investigated whether cerebral toxoplasmosis induced changes in the reactivity of serum toward Toxoplasma gondii excreted-secreted antigens (ESA) in order to develop an assay for evaluating HIV-infected patients with cerebral toxoplasmosis. The antigen selection was based on those produced by tachyzoites, since it is the form of the organism responsible for disseminating the infection, as well as stimulation of the humoral and cellular immune responses. By using an ELISA containing pooled ESA recovered from infected culture supernatants with tachyzoites-RH strain (ESA-ELISA), we found that ESA had a high specificity for sera from patients with cerebral toxoplasmosis. The reactions were compared with an ELISA using crude tachyzoites antigen, widely used in traditional serology. The assays were performed on 293 serum samples separated as follows: 100 sera from patients with cerebral toxoplasmosis and AIDS (symptomatic), 99 sera from individuals with chronic toxoplasmosis (asymptomatic) and 94 sera from healthy individuals without toxoplasmosis (control). The crude tachyzoite antigen in ELISA was able to distinguish both groups of sera with toxoplasmosis, as similar reactivity were observed in sera from patients with cerebral toxoplasmosis and those from chronic individuals. In contrast, ESA-ELISA distinguished sera from symptomatic and asymptomatic individuals (three times more reactive in the former group, 12.6 versus 4.2). The assays were reproducible based on immunoblotting and statistical analysis. These data suggest the utility of ESA-ELISA in the diagnosis of cerebral toxoplasmosis in HIV-infected patients, since it provided clear evidence that anti-ESA antibodies are present principally in patients with active infection. The absence of a significant amount of antibodies distinguished the patients without clinical symptoms of infection.
Journal of Medical Microbiology 08/2008; 57(Pt 7):845-50. · 2.50 Impact Factor
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ABSTRACT: Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and "consensus laboratory criteria" (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis.
Experimental Parasitology 08/2008; 119(3):319-24. · 2.12 Impact Factor
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ABSTRACT: This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.
Experimental Parasitology 03/2008; 118(2):221-7. · 2.12 Impact Factor
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ABSTRACT: Despite highly active anti-retroviral therapy, cryptococcal meningoencephalitis is the second most prevalent neurological disease in Brazilian AIDS patients, being frequently a defining condition with several episodes. As knowledge of Cryptococcus neoformans isolates in the same episode is critical for understanding why some patients develop several episodes, we investigated the genotype characteristics of C. neoformans isolates in two different situations. By pulsed field gel electrophoresis and random amplified polymorphic DNA analysis, 54 isolates from 12 patients with AIDS and cryptococcosis were analyzed. Group 1 comprised 39 isolates from nine patients with a single episode and hospitalization. Group 2 comprised 15 isolates from three patients with two episodes and hospitalizations. Except for three patients from group 1 probably infected with a single C. neoformans isolate, the other nine patients probably were infected with multiple isolates selected in different collection periods, or the infecting isolate might have underwent mutation to adapt and survive the host immune system and/or the antifungal therapy. However, the three patients from group 2 presented genetic diversity among isolates collected in both hospitalizations, possibly having hosted the initial isolate in both periods. These data, emphasize that Cryptococcus diversity in infection can contribute to strategies of treatment and prevention of cryptococcosis.
Memórias do Instituto Oswaldo Cruz 12/2007; 102(7):777-84. · 2.15 Impact Factor
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ABSTRACT: Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding "in vitro" cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.
Veterinary Parasitology 04/2007; 144(3-4):234-41. · 2.58 Impact Factor
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ABSTRACT: Cutaneous leishmaniases present similar clinical appearances, but differing prognosis in the course of infection. Ulcers caused by parasites of the subgenus Viannia are more aggressive than ulcers caused by parasites of the subgenus Leishmania. Another problem is distinguishing between true Leishmania infection and other skin diseases in endemic areas, where cutaneous lesions and a single positive Montenegro intradermal test are enough to submit patients to specific treatment for cutaneous leishmaniasis. This study evaluated the efficacy of PCR in detecting in Leishmania in patients with cutaneous lesions. Leishmania (V.) braziliensis complex was determined by a primer pair from the multicopy spliced leader RNA. The results were compared to those of traditional methods. We analyzed biopsies of 109 patients with cutaneous lesions in the second most endemic region of Sao Paulo State, Brazil. Definitive diagnosis was established by clinical and “consensus laboratory criteria” (positive culture, stained tissue smears or PCR). Of 52 patients with cutaneous leishmaniasis, 96% had positive PCR, 69%, positive parasitological tests and 100%, positive Montenegro intradermal tests. Histopathological examination (only in 32 samples) were positive in 14 samples, suggestive in 14 and negative in 4 samples. All 57 patients with other etiologies had negative results in parasitological methods, PCR and histopathological examination (in 39 samples), but Montenegro intradermal tests were positive in 35%. PCR was highly sensitive and specific for L. (V.) braziliensis complex detection compared with other laboratory methods. Despite the specificity of the parasitological tests, the sensitivity was less than 70%. Montenegro intradermal reaction was highly sensitive, but with low specificity, only 65%. As suggestive results in histopathological examinations were shown in 14 samples, it was difficult to determine the true result. PCR applied to biopsies proved to be useful for differential diagnosis of cutaneous lesions of other etiologies in patients living in endemic areas. The advantages are most striking in clinical specimens with scarce amastigotes for which conventional methods have low sensitivity and should be considered for clinical and epidemiological patterns. On the other hand, both Montenegro intradermal test and parasitological methods are only modestly effective in cutaneous leishmaniasis diagnosis.
Experimental Parasitology.
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ABSTRACT: Despite highly active anti-retroviral therapy, cryptococcal meningoencephalitis is the second most prevalent neurological disease in Brazilian AIDS patients, being frequently a defining condition with several episodes. As knowledge of Cryptococcus neoformans isolates in the same episode is critical for understanding why some patients develop several episodes, we investigated the genotype characteristics of C. neoformans isolates in two different situations. By pulsed field gel electrophoresis and random amplifield polymorphic DNA analysis, 54 isolates from 12 patients with AIDS and cryptococcosis were analyzed. Group 1 comprised 39 isolates from nine patients with a single episode and hospitalization. Group 2 comprised 15 isolates from three patients with two episodes and hospitalizations. Except for three patients from group 1 probably infected with a single C. neoformans isolate, the other nine patients probably were infected with multiple isolates selected in different collection periods, or the infecting isolate might have underwent mutation to adapt and survive the host immune system and/or the antifungal therapy. However, the three patients from group 2 presented genetic diversity among isolates collected in both hospitalizations, possibly having hosted the initial isolate in both periods. These data, emphasize that Cryptococcus diversity in infection can contribute to strategies of treatment and prevention of cryptococcosis.
Memórias do Instituto Oswaldo Cruz (ISSN: 1678-8060) Vol 102 Num 7.
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ABSTRACT: Leishmaniases are endemic in many countries, mainly in rural areas. In Brazil, Leishmania infection is responsible for many cases of Leishmaniases, including recent reports in urban regions. Despite their sensitivity, traditional serological and parasitological methods for detecting Leishmaniases have proven inadequate for species discrimination. This study aimed to identify Leishmania species in biological samples by a fast methodology, avoiding “in vitro” cultivation. Knowledge of the Leishmania species is an important tool in regions where both New World visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are prevalent. As these new foci appear in areas not traditionally endemic for VL, the main problem is to distinguish between true autochthonous infections and infections acquired in other well-known endemic areas. Since, domestic dogs are known to be the main VL and CL reservoir, they are regularly investigated in endemic areas to prevent, principally, severe and often fatal VL in humans. However, several infected dogs present no clinical signs or clinical signs similar to other canine diseases. Here, we evaluated the ability of PCR to diagnose VL and distinguish L. (L.) chagasi from other Leishmania species in domestic dogs. Samples from 114 dogs from 30 cities (Sao Paulo, Brazil) were divided into two groups: 44 symptomatic and 70 asymptomatic. They were assayed by parasitological methods (culture and microscopic examination) and PCR to determine L. (L.) chagasi, L. (V.) braziliensis; and in some cases, Leishmania spp. Parasitological tests and PCR-L. chagasi were concordant in 105 samples (92%). VL was confirmed in 49 dogs, while 56 had negative results. Of the 114 samples, 9 had discordant results, but were further tested by PCR-Leishmania spp. with positive results. VL was also confirmed in 4 dogs having negative parasitological tests and positive PCR-L. chagasi. Consequently, this PCR was positive for 100% (53/49) of dogs with parasites detected in parasitological tests. Also, PCR demonstrated high specificity detecting 61 dogs negative for VL. Leishmania infection was negative in 56 dogs, and 5 with positive culture and PCR-Leishmania spp. had CL since they were positive in PCR-L. braziliensis. This study shows the importance of including PCR in diagnosis of Leishmaniases by differential diagnosis contributing to the surveillance and control of VL programs.
Veterinary Parasitology.
