Genetic variant strains of Leishmania (Viannia) braziliensis exhibit distinct biological behaviors.

A variety of clinical forms of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis, as well as differing immune responses of patients, have been reported for an ACL focus in the state of Minas Gerais, Brazil. In addition, two genetic profiles of L. braziliensis have been described, of which one variant profile (hsp70-variant) has been associated with atypical lesions. We investigated the biological behavior of genetic variant strains of L. braziliensis isolated from patients with different clinical manifestations of ACL. Experimental infections were performed with golden hamsters for five L. braziliensis strains in standardized doses of 1 × 106 parasites per inocula. The characteristics of skin lesions, histopathological features, and parasite burden were independently analyzed at 30 and 60 days post-infection. The data revealed distinct patterns in the onset time of visible skin lesions as well as in lesion size and parasite burden among the strains. The extent and density of the inflammatory infiltrate differed among strains, although cellular composition of granulomas appeared similar. Multivariate analysis indicated the occurrence of two clusters: one comprising native strains (cluster 1) and one comprising the reference strain (cluster 2). Within cluster 1, the genetic variants of L. braziliensis did not group with the non-variant strain suggesting that the distinct patterns of biological behavior of these strains could be associated with the known genetic diversity previously described for them.

Reliability of techniques used in the diagnosis of canine visceral leishmaniasis by the national control program in Brazil: A survey in an area of recent transmission.

One of the key components of the Brazilian Program for the Control of Visceral Leishmaniasis (PCLV) is the euthanasia of Leishmania-infected canine reservoirs, the detection of which depends on a screening procedure involving a Dual Path Platform® (DPP) immunoassay and a confirmatory enzyme-linked immunosorbent assay (ELISA). The aims of the present study were to evaluate the reliability of these techniques in a region of recent transmission of canine VL, to follow up the seroconversion 3-4 months after the initial diagnosis of DPP reactive but ELISA indeterminate or non-reactive dogs, and to identify the species of Leishmania in circulation in the area. Each animal was submitted to DPP under field conditions, performed by municipal health workers using peripheral blood (DPP-field), to DPP under laboratory conditions using serum (DPP-lab) and to ELISA using serum. The agreements between the tests were determined using McNemar's χ2 test, Cohen's kappa coefficient (k) at the 95% confidence interval and prevalence-adjusted bias-adjusted kappa (PABAK). Of the 1130 dogs examined, 74.2% were non-reactive in all three tests applied. Based on the PCLV positive-infection criterion, seroprevalence was 8.9% (101/1130) with 83.2% (84/101) of infected animals showing reactivity in all three tests while 7.8% (8/101) were reactive in DPP-field and ELISA and 8.9% (9/101) in DPP-lab and ELISA. The proportions of disagreements were substantial in all comparisons. Inter-rater reliability between DPP-field and ELISA (k=0.55; PABAK=0.78) and DPP-lab and ELISA (k=0.59; PABAK=0.81) were considered moderate, while that between DPP-field and DPP-lab (k=0.61; PABAK=0.79) was classified as marginally good. The proportion of seroconversions in DPP reactive animals that were initially ELISA indeterminate was significantly higher than in those that were DPP reactive but initially ELISA non-reactive. Restriction fragment length polymorphism analysis revealed the presence of Leishmania infantum, the etiologic agent of VL, in bone marrow samples from VL-infected animals. Our data showed that the techniques and protocols currently employed in the PCLV screening approach are not entirely reliable. Further consideration should be given to monitoring dogs with undetermined results in ELISA and a better training should be provided for health workers responsible for performing DPP tests applied under field conditions.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil.

INTRODUCTION: Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. METHODS: We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. RESULTS: The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. CONCLUSIONS: The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals. .

Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis.

The vectorial capacity of Rhipicephalus sanguineus in the transmission of canine visceral leishmaniasis has been evaluated through a laboratory-controlled experiment. One healthy Leishmania-free dog and two dogs naturally infected with Leishmania were infested with R. sanguineus in various stages of development. Engorged larvae, unfed nymphs, engorged nymphs, unfed adults, engorged female adults and fed male adults were collected from the experimental animals and examined for Leishmania infection by optical microscopy, polymerase chain reaction (PCR) and parasite culture. Leishmania forms were not detected in any of the 433 smears prepared from engorged colonies nor in any of the 118 smears prepared from unfed colonies. However, one flagellate structure was identified in one of the smears. All pools of R. sanguineus that had fed on the infected dogs tested PCR-positive for Leishmania DNA, with the single exception of the pool of engorged larvae. In contrast, all pools of ticks that had fed on the Leishmania-free dog were PCR-negative. Leishmania growth was not observed in any of the tick colonies following incubation on culture medium. Considering that no Leishmania forms were identified in any of the meticulously analysed smears derived from engorged colonies of R. sanguineus, it appears somewhat unlikely that the maintenance and multiplication of Leishmania occurs within the tick.

Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies.

Visceral leishmaniasis (VL) is a growing zoonosis with an increasing number of new cases and a rapid geographical spreading of the disease. In the present study, a canine survey was carried out in the city of Montes Claros (320,000 inhabitants), an endemic area of American visceral leishmaniasis in the state of Minas Gerais, Brazil. A total number of 4795 dogs were examined by serology, which showed a rate of seropositivity of 5%. Isoenzymatic analysis confirmed Leishmania infantum chagasi as the local aetiological agent of CVL. Canine tissues were assayed for the presence of Leishmania parasite DNA using different techniques. The infectivity of asymptomatic, oligosymptomatic and symptomatic seropositive dogs was tested by xenodiagnosis using laboratory reared Lutzomyia longipalpis. Rates of infection of 5.4%, 5.1% and 28.4% were found for the phlebotomine sand flies that fed in asymptomatic, oligosymptomatic and symptomatic dogs, respectively. Our results indicate that, under experimental conditions, symptomatic dogs are about four times more infective to VL vectors than oligosymptomatic or asymptomatic animals. The lower infectivity rates of dogs displaying any of the last two clinical forms of leishmaniasis, however, must be taken into account in the epidemiology of CVL.