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Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and Allopurinol
Abstract
A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.
... However, the animals did not give a negative specific antibody response at the end of the follow-up, at 300 days after treatment. Our results are consistent with those obtained by others authors who described a decrease in the levels of anti-Leishmania antibodies after treatments and dogs did not present negative titers at the end of treatment (Ribeiro et al., 2008;Manna et al., 2008Manna et al., , 2015Andrade et al., 2011;Cantos-Barreda et al., 2018). ...
... However, the 7-day drug regimen did not achieve parasite eradication as all 6 dogs gave a positive parasitological culture test and/or PCR test at 10 months post therapy for lymph node, spleen and/or bone marrow samples. This same inability to produce a complete parasitological cure is shown by other drugs and combinations of drugs evaluated so far (Schettini et al., 2005;Ribeiro et al., 2008;Vexenat et al., 1998;Da Silva et al., 2012;Athanasiou et al., 2013Athanasiou et al., , 2014Manna et al., 2008Manna et al., , 2015Andrade et al., 2011;Dos Santos Nogueira et al., 2019). However, joint treatment was as effective or even better than other reported treatment protocols. ...
... However, joint treatment was as effective or even better than other reported treatment protocols. This includes treatment with PMM solution (15-30 days, Vexenat et al., 1998;Athanasiou et al., 2013Athanasiou et al., , 2014, antimonial drug solution (45 days, Poli et al., 1997), combination treatment with MA and allopurinol for 30 days (Manna et al., 2008(Manna et al., , 2015, combination treatment with MA and PMM for 21days (Oliva et al., 1998), PMM and allopurinol for 28 days (Kasabalis et al., 2019(Kasabalis et al., , 2020, administration of liposomal MA (4 days, Schettini et al., 2005;Ribeiro et al., 2008), oral administration with miltefosine for 28-45 days (Andrade et al., 2011;Dos Santos Nogueira et al., 2019), combination treatment of miltefosine and alopurinol (30 days, Manna et al., 2015) or oral administration of a new alkylphosphocholine molecules (Hernández et al., 2014;Hernández Martínez, 2015). Therefore, increasing the dose drug administered or giving a second round of SSG-NIV: PMM-NIV treatment could result in parasite eradication or prevent relapse of the treated dogs. ...
Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.
... Current therapeutic measures to decrease infection in dogs and the transmission to humans are often cost-prohibitive and/or not efficacious. 6,[35][36][37][38][39][40][41] Treatment of CanL is difficult or even prohibited in some countries. 37,[40][41][42][43] This is due to the fear of the development of drug-resistant strains of Leishmania based on veterinary use as the same drugs are used to treat disease in dogs and humans. ...
... 6,[35][36][37][38][39][40][41] Treatment of CanL is difficult or even prohibited in some countries. 37,[40][41][42][43] This is due to the fear of the development of drug-resistant strains of Leishmania based on veterinary use as the same drugs are used to treat disease in dogs and humans. 37,[40][41][42][43] The most common treatment of choice for VL in humans worldwide is amphotericin B (liposomal or conventional) with or without the combination of pentavalent antimony. ...
... 37,[40][41][42][43] This is due to the fear of the development of drug-resistant strains of Leishmania based on veterinary use as the same drugs are used to treat disease in dogs and humans. 37,[40][41][42][43] The most common treatment of choice for VL in humans worldwide is amphotericin B (liposomal or conventional) with or without the combination of pentavalent antimony. 44 However, these therapies are often not well tolerated, are cost-prohibitive, and have become complicated by antimonial-resistant Leishmania strains in Other therapeutic options include miltefosine and paromomycin, which either have not been tested for efficacy against visceralizing forms of infection or have a lower efficacy than aforementioned treatments. ...
InLeishmania infantum-endemic countries, controlling infection within dogs, the domestic reservoir, is critical to public health. There is a need for safe vaccines that prevent canine progression with disease and transmission to others. Protective vaccination againstLeishmaniarequires mounting a strong, inflammatory, Type 1 response. Three commercially available canine vaccines on the global veterinary market use saponin or inflammatory antigen components (Letifend) as a strong pro-inflammatory adjuvant. There is very little information detailing safety of saponin as an adjuvant in field trials. Safety analyses for the use of vaccine as an immunotherapeutic in asymptomatically infected animals are completely lacking.Leishmania infantum, the causative agent of canine leishmaniasis, is enzootic within U.S. hunting hounds. We assessed the safety of LeishTec®after use in dogs from two different clinical states: 1) without clinical signs and tested negative on polymerase chain reaction and serology or 2) without clinical signs and positive for at least oneLeishmaniadiagnostic test. Vaccine safety was assessed after all three vaccinations to quantify the number and severity of adverse events. Vaccinated animals had an adverse event rate of 3.09%, whereas placebo animals had 0.68%. Receiving vaccine was correlated with the occurrence of mild, site-specific, reactions. Occurrence of severe adverse events was not associated with having received vaccine. Infected, asymptomatic animals did not have a higher rate of adverse events. Use of vaccination is, therefore, likely to be safe in infected, asymptomatic animals.
... Consistent with previous studies, we detected a significant decline in the blood parasite load during the first 30 days of treatment and a maintenance of reduced parasitemia through all the treatment period (Baneth and Shaw, 2002;Manna et al., 2008a;Martinez et al., 2011;Solano-Gallego et al., 2016a). However, levels of parasitemia were detected along treatment follow-up even though at low levels in comparison with other studies (Manna et al., 2008a;Manna et al., 2008c). Leishmania infantum-infected dogs, whose blood, lymph nodes and skin samples were screened by RT-PCR showed that during a long period of treatment, a progressive decrease of parasite load was associated with clinical recovery, even though a marked parasite load was still present in lymph nodes in 50% of dogs after 24 months of treatment (Manna et al., 2008c). ...
... However, levels of parasitemia were detected along treatment follow-up even though at low levels in comparison with other studies (Manna et al., 2008a;Manna et al., 2008c). Leishmania infantum-infected dogs, whose blood, lymph nodes and skin samples were screened by RT-PCR showed that during a long period of treatment, a progressive decrease of parasite load was associated with clinical recovery, even though a marked parasite load was still present in lymph nodes in 50% of dogs after 24 months of treatment (Manna et al., 2008c). Additionally, in our study, presence of L. infantum DNA was detected in some dogs after negative RT-PCR results were obtained in preceding follow-up samples. ...
... Therefore, blood RT-PCR during treatment monitoring should be accompanied by a full physical examination, quantitative serology and routine laboratory tests. Importantly, in our study the majority of treated dogs achieved a clinical cure while parasitological load did not completely vanish as previously reported (Manna et al., 2008c). ...
There is limited data regarding Leishmania infantum specific T cell mediated immunity in naturally infected sick dogs at the time of diagnosis and during anti-Leishmania treatment. Our aim was to investigate the kinetics of L. infantum specific IFN-γ production in dogs with leishmaniosis at the time of diagnosis and during treatment and to correlate it with specific L. infantum antibodies, blood parasitemia and clinicopathological findings. Thirty-four dogs were diagnosed with leishmaniosis based on physical examination, routine laboratory tests and L. infantum-specific antibody levels by quantitative ELISA. Heparinized whole blood was stimulated with L. infantum soluble antigen (LSA) and concanavalin A (ConA) and incubated for 5 days. IFN-γ concentration was evaluated in supernatants of stimulated blood using a commercial sandwich ELISA. Leishmania real-time PCR was also performed for assessing blood parasitemia. Dogs were treated with meglumine antimoniate and allopurinol. Sixteen dogs were classified as IFN-γ non-producers after LSA stimulation (mean ± SD: 0 ± 0 pg/mL) and 18 dogs as IFN-γ producers (mean ± SD: 2885.3 ± 4436.1 pg/mL) at the time of diagnosis (P < 0.0001). IFN-γ non-producers were classified in a more severe clinical staging than IFN-γ producers that presented a mild to moderate clinical staging (P = 0.03). In the IFN-γ non-producer group, production of IFN-γ after LSA stimulation was significantly increased during treatment especially at day 365 (P = 0.018) together with clinical improvement when compared with day 0. In contrast, IFN-γ producers maintained their IFN-γ production after LSA stimulation and no statistically significant changes were found during treatment follow-up. At diagnosis, IFN-γ non-producers showed a significantly higher blood parasitemia versus IFN-γ –producers (P = 0.005). IFN-γ non-producers drastically reduced blood parasitemia to minimum values at day 365 when compared with day 0 (P = 0.017). No significant differences were found at day 365 in blood parasitemia of IFN-γ producers compared to pre-treatment. At diagnosis, L. infantum specific antibodies were higher in IFN-γ non-producers than IFN-γ producers (P = 0.014). A marked reduction of antibody levels was found at day 365 when compared with day 0 in IFN-γ non-producers (P = 0.005) and producers (P = 0.001). These results demonstrate that IFN-γ concentration increases with long-term anti-Leishmania treatment together with clinical improvement in dogs that do not produce IFN-γ at diagnosis. Together with clinical recovery, reduction in blood parasitemia and L. infantum specific antibodies, tracking IFN-γ concentration could constitute an important prognostic tool for immune monitoring in CanL.
... Long term allopurinol treatment, whether combined with an initial course of meglumine antimoniate or miltefosine [8,10,11], or administered alone [12,13], results in most cases in improvement of the dog's clinical disease signs within a few weeks and a reduction in the dog's infective potential. Despite that, cases of disease relapse following secession of combined or exclusive allopurinol treatment [14,15,16] or even during treatment [17,18] have been recorded. Despite a growing concern over the development of resistance to other antileishmanial drugs in humans and increased efforts to uncover the genetic and biochemical basis of resistance mechanisms [19,20,21,22,23,24], very little information is available on resistance to anti-leishmanial drugs in dogs and no direct association has been documented between disease relapse and drug resistance [25,26,27]. ...
... The clinical score was able to reliably reflect the clinical status of each group, while serology, complete blood count results, as well as the biochemical indices tested, did not show significant differences between groups that could be beneficial for their clinical characterization. A previous study has demonstrated that an increase in parasite DNA loads in lymph nodes, but not blood, was correlated with the reappearance of clinical signs in relapsing dogs [17]. Our results shows that parasitemia seems to be greatly reduced or absent following treatment, which may be attributed to the effect of allopurinol, causing a total reduction in parasite load, as reported previously [35]. ...
... Thus, these results do not indicate that any ancillary test employed in the study is superior to the clinical score in the diagnosis of relapse in leishmaniotic dogs. Although it is widely accepted that dogs treated for visceral leishmaniasis using the currently available drugs frequently remain infected and often relapse [8,13], only a small number of relapse cases have been described so far [14,15,16,17,18], and no characterization of parasite strains from relapsed dogs was made. As opposed to the large volume of evidence on drug resistance in human leishmaniasis, little is known regarding the development of drug resistance in CanL. ...
Background:
Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of Leishmania strains from dogs to allopurinol has not been described before in clinical studies.
Methodology/principal findings:
Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group) showed an average half maximal inhibitory concentration (IC50) of 996 μg/mL. A significantly lower IC50 (P = 0.01) was found for isolates from ten dogs before treatment (NT group, 200 μg/mL), as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL). Axenic amastigotes produced from isolates of the TR group also showed significantly higher (P = 0.002) IC50 compared to the NT group (1678 and 671 μg/mL, respectively). The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (P = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol).
Conclusions:
This is the first study to demonstrate allopurinol resistance in L. infantum and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.
... Assays based on the detection of kinetoplast DNA (kDNA) appear to be the most sensitive for direct detection in infected tissues [49,50]. Real-time PCR allows quantification of the Leishmania parasite load in the tissues of infected dogs, which is useful for the diagnosis and the follow-up during treatment [51,52]. It is important to highlight that information provided by PCR should not be separated from the data obtained from clinicopathological and serological evaluations. ...
... Dogs with renal insufficiency are expected to have a lower recovery rate in comparison to those without kidney compromise or only mild proteinuria. Therapy with antileishmanial drugs often leads to clinical cure [54] although treated dogs may continue to harbour the parasite and be infectious to sand flies, but to a lesser extent than pre-treatment [52,[55][56][57]. ...
... The vast majority of dogs experience clinical improvement within the first month of therapy [51,52,58]; however, a longer period of therapy may be required for others before improvement is apparent. The frequencies of monitoring and clinicopathological parameters, including serology, to be followed up during treatment of CanL are summarized in Table 6. ...
... Accordingly, we found that dogs living outdoors tend to frequently develop clinical signs of CanL, while it is more likely that the infection remains subclinical in dogs kept indoors. Fur-thermore, the regression model confirmed that living outdoors represents a significant risk factor for progression to severe stages (III and IV) of CanL, probably due to a higher parasite load linked to a longer exposure to the vector (Pennisi et al., 2005;Manna et al., 2008;Muniesa et al., 2016). Nevertheless, the use of topical pyrethroids with proven repellent effects against sandflies, which are also reported to be one of the most useful and effective way in preventing L. infantum infection in dogs (Otranto and Dantas-Torres, 2013), was not associated with a reduction in the severity of the disease, despite being widely used among the CanL group (67.2%). ...
... Therefore we conducted three XP-EHH analyses to identify signals that may reflect the different risks for developing severe forms of CanL (stages III and IV) observed between clinically susceptible and clinically resistant breeds (Table 4). Clinically susceptible breeds that significantly correlated with outdoors habitat were excluded from the genomic analyses, as their predisposition could be strongly affected by a longer exposure to the vector and other environmental factors, apart from genetics (Ming and Davis, 2003;Pennisi et al., 2005;Manna et al., 2008;Quiñonez-Díaz et al., 2012;Muniesa et al., 2016;Vialard and Olivier, 2020;Hirakawa et al., 2020). In contrast, breeds that were not linked with an outdoor habitat are more likely to display susceptible genotypes for severe forms of CanL. ...
... The most common treatment consists on antimonials, which actively reduce parasitic load, together with allopurinol, which has a parasitostatic effect and, therefore, maintains parasitic load at low levels [2,5,11]. These drugs can cause significant adverse effects, most frequently nephrotoxicity [12,13], urolithiasis and crystalluria [14,15] or digestive disorders [16]. In addition, resistances to several of these drugs have also been documented such as resistances to antimonials [17] or allopurinol [18]. ...
... Further studies must investigate the use of domperidone in dogs with clinical leishmaniosis in combination with traditional therapies such as antimonials and allopurinol. The use of immunotherapy could shorten treatment duration, which could reduce the incidence of adverse effects produced by traditional therapies and also improve the prognosis [12,13,16,19]. Furthermore, studies on the effect of domperidone on transmissibility of L. infantum could be an interesting area of future research as seropositive healthy dogs (subclinical dogs) are an important consideration for public health [69,70]. ...
Background
Domperidone (Leisguard®) is an immunomodulatory drug used as a preventive measure in healthy dogs. However, no studies have been published in healthy Leishmania infantum-seropositive dogs. The aim of this study was to evaluate the clinical efficacy and safety of domperidone as immunotherapy in Leishmania-seropositive healthy dogs.
Methods
Sixty-seven dogs were treated with domperidone at 0.5 mg/kg and 44 dogs received placebo, once daily for 4 consecutive weeks. Monthly treatments were repeated every 4 months until the end of the 1-year follow-up period. Veterinary examinations were performed on days 0, 30, 120, 150, 240, 270 and 360. Samples of blood and urine were collected on days 0, 120, 240 and 360 for routine laboratory tests and quantitative in-house ELISA for the detection of L. infantum-specific antibodies. Furthermore, Leishmania real-time PCR and IFN-γ ELISA were performed at day 0 and the end of the study. Dogs that developed disease were withdrawn from the study and classified as sick dogs. Adverse drug reactions were reported.
Results
Thirty dogs developed disease during the follow-up period: 13/67 (19.4%) in the group treated with domperidone and 17/44 (38.6%) in the placebo-treated group (P = 0.03). Low-seropositive dogs treated with domperidone (4/40, 9.1%) were significantly less likely to develop disease compared to low-seropositive dogs treated with placebo (7/24, 29.2%; P = 0.04), while no differences were found between domperidone (9/23, 39.1%) and placebo (10/20, 50%) in medium- to high-seropositive dogs. At the end of the study, a higher proportion of Leishmania PCR-positive dogs was observed in the placebo-treated group (16/33, 48.5%) compared to the domperidone group (13/51, 25.5%; P = 0.04). Furthermore, low-seropositive dogs treated with domperidone with an increase of IFN-γ concentration presented a higher increase than those treated with placebo at the end of the study. Four dogs treated with domperidone presented self-limiting diarrhea.
Conclusions
Healthy dogs with low L. infantum antibody levels treated with domperidone were less likely to develop disease compared to placebo-treated dogs. Furthermore, domperidone presented a good safety profile.
Graphical abstract
... In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116,118]. ...
... In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116,118]. ...
There is a possibility that during a pet's lifetime, medication may be recommended to treat medical conditions or problems. This book Canine Medicine - Recent Topics and Advanced Research provides the knowledge in diagnosis and treatment of some important diseases and problems that the canines face. I believe that this book offers broader perspective to the readers in the recent advances in canine medicine, starting from recent topics to application in clinical diagnosis and therapeutics for practitioners and veterinarians. The main purpose of the book is to point out the interest of some important topics of canine medicine and the progress in this field and to clear its importance in veterinary medicine.
... In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116,118]. ...
... In accordance with Ikeda-Garcia et al. [116], Manna et al. [118] monitored leishmania DNA load in infected dogs through real-time PCR, using a similar treatment protocol. Therapy resulted in clinical improvement accompanied by reduction in parasite burden, but even after a long period of treatment with allopurinol alone, parasites remained in tissues [116,118]. ...
Visceral leishmaniasis (VL) is a disease caused by a protozoon belonging to the genus Leishmania, and it is transmitted through the bite of sand flies. Endemic regions have widened, and canine visceral leishmaniasis (CVL) occurs mainly in the Mediterranean region and South America. There is no consensus on the risk factors associated with CVL, as results differ between the studied regions and countries. This chapter describes the main aspects of epidemiology, immunology, clinical signs, diagnosis treatment, and control of canine visceral leishmaniasis with emphasis on Brazil.
... Tissue DNA Kit (Omega Biotech VWR) according to the manufacturer's instructions. The PCR test was targeted on a 123 bp fragment inner the constant region in the minicircle kinetoplast DNA (NCBI accession number AF291093) and was carried out as previously described [11]. The primers and probe were chosen with the assistance of Primer Express Software (Applied Biosystems). ...
... This suggests that cytology is useful in these lesions but also that inflammation of the conjunctiva and cornea could be directly related to the protozoan rather than to an altered immune response due to infection [6]. The granulomas on the eyelid margin could also be a reaction at the vector biting site, as hypothesized by other researchers [6,11]. ...
Aim:
The point prevalence of ocular lesions due to leishmaniasis was evaluated in 127 dogs living in a municipal shelter placed in a highly endemic area (Sicily, Italy). Moreover, the period prevalence, the type, and prognosis of lesions due to leishmaniasis were evaluated in 132 dogs with ocular pathologies referred to a Veterinary Teaching Hospital (VTH) in the same endemic area over a 3-year period.
Materials and methods:
All the dogs were submitted to ophthalmological examination. The diagnosis of leishmaniasis was made by cytological, serological (immune-fluorescent antibody test), and molecular (quantitative polymerase chain reaction) tests.
Results:
The point prevalence of ocular lesions in 45 shelter dogs with leishmaniasis was 71.11% (45/127 dogs). The most frequent ocular lesion was blepharitis (50%) while anterior uveitis was observed in only 9.37% of cases. The period prevalence of ocular lesions due to leishmaniasis in the VTH group was 36.36% (48/132 dogs). In both groups, most of the lesions were bilateral and involved the anterior segment. Anterior uveitis was the most frequent ophthalmic finding in client-owned dogs (37.50%), but it occurred in only 9.37% of the shelter dogs. Keratouveitis often occurred during or after antiprotozoal treatment (14.58%; 7/48). In this study, the healing of eye injury following systemic antiprotozoal treatment was recorded in about half of cases (48%; 12/25 dogs), in which follow-up was possible. In more than 1/3 of cases (36%; 9/25), there was an improvement, but it was necessary to associate a long-term topical treatment; most of them, as well as those who had not responded to systemic therapy (16%; 4/25), had anterior uveitis or keratoconjunctivitis sicca.
Conclusions:
Ocular manifestations involve up to 2/3 of animals affected by canine leishmaniasis and lesions account for over 1/3 of ophthalmic pathologies observed at a referral clinic in an endemic area. The occurrence of anterior uveitis is more frequent in client-owned dogs than in shelter dogs. The onset of keratouveitis during or after antiprotozoal treatment could be attributed to the treatment or to a recurrence of the systemic form. The post-treatment uveal immune reaction, already observed in humans, could explain the difference in the frequency of keratouveitis between client-owned and shelter dogs, which have never been treated.
... We observed a significant decline in the blood parasite load during the first 30 days of treatment and a progressive reduction throughout the remainder of the period, consistent with previous studies [12,13,30]. However, as previously described [32,33], after long periods of treatment, low levels of parasitemia might be observed. The presence of L. infantum DNA was detected in some dogs in the present study after negative follow-ups. ...
... Therefore, blood real-time PCR during treatment monitoring should always be accompanied by a full physical examination, quantitative serology and routine laboratory tests. In addition, it is important to point out that in the majority of these treated dogs, clinical cure exists while parasitological cure does not as previously reported [32,33]. It is also likely that long term treatment with allopurinol will induce parasite drug resistance as elegantly documented in natural clinical leishmaniosis in dogs [26]. ...
Background
Leishmania infantum-specific antibodies are used extensively for the diagnosis and monitoring of treatment in canine leishmaniosis. Different views have been described for the measurement of L. infantum antibody levels for the monitoring of anti-leishmanial treatment. In addition, molecular techniques using blood are frequently employed in the clinical setting. However, there are not enough studies to prove the usefulness of PCR in diagnosis, treatment monitoring and in assessing the prognosis of the disease. The objectives of this study were to evaluate L. infantum-specific antibodies and blood parasitemia at the time of diagnosis and during treatment and to correlate these with the dog’s clinical status.
Methods
Thirty-seven dogs were diagnosed and followed-up during treatment (days 30, 180 and 365). The treatment protocol consisted of a combination of meglumine antimoniate for one month and allopurinol for at least one year. Leishmania infantum-specific antibodies and blood parasitemia were assessed by an end point sera dilution ELISA and by real-time PCR, respectively.
Results
The majority of dogs were classified as LeishVet stage II (moderate disease) at the time of diagnosis (86 %) and the rest as stage III. Results showed variable levels of specific antibodies at the time of diagnosis [median ± interquartile range (IQR): 1372 ± 8803 ELISA units (EU)]. Twenty-three seropositive dogs (64 %) were detected as PCR-positive at the time of diagnosis. Interestingly, a rapid significant antibody level reduction was observed by day 30 of treatment (median ± IQR: 604 ± 2168 EU). A continuing significant decrease of specific antibodies was also found at days 180 (median ± IQR: 201 ± 676 EU) and 365 (median ± IQR: 133 ± 329 EU) in association with clinical improvement. A significant blood parasitemia reduction was also observed at all time points studied. Mean parasites/ml ± SD were 19.4 ± 79.1 on day 0, 2.2 ± 11.7 on day 30, 0.9 ± 2.9 on day 180, and 0.3 ± 0.7 on day 365.
Conclusions
This study reports a significant reduction of L. infantum antibodies measured by an end point sera dilution ELISA method after 30 days of treatment associated with clinical improvement. A low proportion of sick dogs with moderate disease were negative by blood real-time PCR at the time of diagnosis.
... This method helps to monitor the parasite load in different tissues throughout the course of the disease and after treatment [72]. It presents the disadvantage that it is expensive and requires qualified personnel for interpretation. ...
Laboratory diagnosis of leishmaniasis remains a major challenge, demonstrating variable efficacy in detecting infected mammalian hosts. Thus, there is a great need for the accuracy of the identification of appropriate antigens to also improve diagnostic tests. This review brings to the fore the sensitivity of the balance in canine and human leishmaniasis, which greatly influences the progression of the disease and especially the diagnostic methods. The problem of diagnosis arises in asymptomatic leishmaniasis, since in these conditions the methods considered as reference in the diagnosis of leishmaniasis no longer show certainty, being influenced for the most part by the immune response of the host, which is different according to the presence of other associated diseases or even according to the breed, when we talk about dogs. Consequently, the diagnosis and surveillance of leishmaniasis cases remains an open topic, with the need for new methods adapted to the immunological status of the host.
... The most accurate and precise way to diagnose Leishmania is PCR from the bone marrow, lymph nodes, spleen, or skin [36][37][38]. Whole blood, buffy coat, and urine have a much lower PCR sensitivity than the previously stated tissues [36,39]. The two biggest risk factors for dogs getting leishmaniasis are lifestyle and exposure to sandflies [40]. ...
Canine vector-borne diseases are of great relevance not only regarding animal welfare but also in relation to the One Health concept. Knowledge concerning the most relevant vector-borne pathogens in dogs is scarce and limited to stray dogs in most western African regions, and there is virtually no information about the situation in kept dogs presenting (regularly) to vets. Therefore, the blood samples of 150 owned guard dogs in the Ibadan area—in the southwest of Nigeria—were collected and analyzed for the DNA of Piroplasmida (Babesia, Hepatozoon, Theileria), Filarioidea (e.g., Dirofilaria immitis, Dirofilaria repens), Anaplasmataceae (e.g., Anaplasma, Ehrlichia), Trypanosomatidae (e.g., Leishmania, Trypanosoma), Rickettsia, Bartonella, Borrelia and hemotropic Mycoplasma using molecular methods. Overall, samples from 18 dogs (12%) tested positive for at least one pathogen. Hepatozoon canis (6%) was the most prevalent blood parasite, followed by Babesia rossi (4%). There was a single positive sample each for Babesia vogeli (0.6%) and Anaplasma platys (0.6%). Moreover, one mixed infection with Trypanosoma brucei/evansi and Trypanosoma congolense kilifi was confirmed (0.67%). Generally, the prevalence of vector-borne pathogens in this sample group of owned dogs in southwest Nigeria was lower than in prior studies from the country and in other parts of Africa in total. This leads to the assumption that, firstly, the exact geographical location has a major influence on the incidence of vector-borne diseases, and, secondly, it seems to make a difference if the dogs are owned and, therefore, regularly checked at a veterinary clinic. This study should raise awareness of the importance of routine health check-ups, tick and mosquito prophylaxis, and a well-managed infectious disease control program to prevent vector-borne diseases in canines.
... Researchers, clinical laboratories, and insurers choose laboratory test requirements to maximize the relationship between sensitivity, specificity, simplicity and cost-benefits (22)(23)(24)(25). Molecular techniques could play an important role in detection and the monitoring of therapy in humans and animals leishmaniasis (26,27). Based on our study, the three molecular methods confirmed that all 50 positive sam-ples belong to L. tropica species. ...
Background: To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. Methods: This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The posi-tives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. Results: All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. Conclusion: HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity , cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.
... Researchers, clinical laboratories, and insurers choose laboratory test requirements to maximize the relationship between sensitivity, specificity, simplicity and cost-benefits (22)(23)(24)(25). Molecular techniques could play an important role in detection and the monitoring of therapy in humans and animals leishmaniasis (26,27). Based on our study, the three molecular methods confirmed that all 50 positive sam-ples belong to L. tropica species. ...
Background: To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches. Methods: This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The posi-tives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays. Results: All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases. Conclusion: HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity , cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.
... Além disso, existe uma preocupação com a resistência às drogas leishmanicidas utilizadas no tratamento humano que limita o uso dessas drogas no tratamento canino. Cães tratados permanecem infectados por um longo período, pois os medicamentos utilizados apenas prolongam a vida do animal e melhoram sua condição clínica, com altas chances de recorrência dos sinais clínicos (Andrade et al., 2011;Manna et al., 2008;Woerly et al., 2009;Yasur-Landau et al., 2016). infantum dentro de fagócitos em mafíferos infectados (C1) e transmite a forma promastigota durante o repasto sanguíneo (B1). ...
O objetivo desta revisão é esclarecer o triplo papel, reservatório-vítima-sentinela, que o cão exerce na leishmaniose visceral e discutir o foco na eliminação do “melhor amigo do homem” para proteção do ecossistema, levando-se em conta os três pilares epidemiológicos, parasito-vetor-hospedeiro, e os aspectos éticos. Logo, inicialmente serão abordados aspectos epidemiológicos relacionados a Leishmaniose Visceral (LV) e a Leishmania Visceral Canina (LVC). Posteriormente, uma reflexão sobre o papel do cão neste contexto, tido como principal reservatório da Leishmania infantum no ambiente urbano, será realizada visando a compreensão de medidas como a eutanásia, de animais positivos; a manutenção do cão parasitado na família (vítima) e o uso destes animais como sentinelas para a detecção da LV e LVC. Finalmente, conclui-se que a LVC é um problema que aflige a família multiespécie brasileira, afetando seres humanos e cães. Os métodos de controle e vigilância paras a LV implementadas no Brasil ainda são ineficientes e desatualizados, desconsiderando o cão como membro da família e tratando-o unicamente como hospedeiro reservatório. Portanto é fundamental uma revisão no Programa de Controle e Vigilância que inclua a Saúde Única na forma de lidar com as leishmanioses: hospedeiros, vetores e parasitos, dentro de um mesmo ambiente para dirigir recursos em medidas eficientes, com o cão sendo utilizado como sentinela da LV.
... Our findings suggest that the hematogenous dissemination occurs only in a small number of sick animals, confirming that the routine screening of blood samples, through PCR and smear microscopy, must not be currently advised to establish a diagnosis, because of lower diagnostic sensitivity compared with bone marrow or lymph node samples (Sharma et al. 2000;Delgado et al. 1998;Oikonomidis et al. 2019); the sensitivity increases performing a buffy coat (Oikonomidis et al. 2019). Furthermore, the blood parasite load was very low even in dogs with high lymph node parasite loads and high IFAT titers, in accordance with other reports describing natural infections (Manna et al. 2008;. The lack of correspondence between lymph node and blood had already been observed in previous studies performed with microscopic methods in humans affected by Kala-azar due to Leishmania donovani (Sharma et al. 2000;Saran et al. 1997). ...
The aim of this study was to evaluate, through qPCR, the prevalence of parasitemia in sick kennel dogs naturally infected by canine leishmaniasis. An evaluation of daily changes of the parasitic load in peripheral blood was also performed. A comprehensive clinical examination and the collection of several samples (blood, lymph node, skin, and conjunctiva) were performed in 140 dogs living in an endemic area. Among these, only the dogs with clinically evident leishmaniasis were enrolled (39/140; 27.9%). Twelve (30.8%) out of 39 showed parasitemia, with a low load (median: 4 Leishmania/ml) despite a high lymph node parasite load (median: 4000 Leishmania/ml) and high IFAT titers (≥ 1:640). Seven sick dogs were sampled every 4 h for 6 times during a 24-h period, in order to obtain light- and dark-span samples. Only one (14.3%) out of the seven serial sampled dogs showed Leishmania DNA in the peripheral blood in two samples (2/42; 4.8%). Surprisingly, Leishmania DNA was also detected in the peripheral blood of asymptomatic dogs, negative to both serology and PCR performed on samples other than blood (6/101; 5.9%). The present study confirms that in canine leishmaniasis parasitemia is uncommon and even transitory. Even if recommended, microscopic examination is confirmed as a low sensitivity method with a lower diagnostic utility in canine leishmaniasis than qPCR. Moreover, circulating Leishmania DNA can be found even in healthy dogs. This finding is important in clinical practice because in endemic areas it suggests a transfusion risk and a possible transmission to the vector.
... RT-PCR is fast and has an extensive dynamic range as there is no requirement of opening the reaction tube; also, cross-contamination is decreased. RT-PCR is convenient for diagnosis of PKDL (Ghosh et al. 2018) and canine leishmaniasis where it helps in monitoring parasitic load in different tissues throughout and after the treatment (Manna et al. 2008;Pennisi et al. 2005). ...
Diagnosis of leishmaniasis has always been a major challenge as its clinical features resemble some other commonly occurring diseases such as tuberculosis, typhoid, and malaria. Reliable laboratory methods become important for differential diagnosis. Demonstration of the parasites in stained preparations of bone marrow and splenic aspirates being risky and invasive is still the gold standard for diagnosis. Serological tests utilizing rapid immunochromatographic formats or rK39 in enzyme linked immune sorbent assay, immunoblotting, direct agglutination test have complications related to high proportions of positive asymptomatic individuals and the inability to diagnose a relapse. Among the molecular techniques, polymerase chain reaction is the most commonly used technique that is successfully implied for diagnosis. This review provides updated information on the recent developments in the field of diagnosis in leishmaniasis, various methods utilized with their advantages and limitations.
... The qPCR sensitivity varied between peripheral blood (38.5%) and bone marrow (95.6%). The decreased sensitivity observed in blood samples could be due to the low parasite load in the peripheral blood compared to bone marrow or lymph node (Manna et al., 2008;Solano-Gallego et al., 2007) along with the low amount of template DNA, due to amplification of DNA from a punch of filter paper. ...
Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.
... Thus, the most frequent treatment is usually a combination of antimonials or miltefosine with allopurinol, which maintains the parasitic load at low levels (Reguera et al., 2016;Solano-Gallego et al., 2011). However, conventional anti-Leishmania drugs used in dogs can induce side effects such as nephrotoxicity, urolithiasis and digestive disorders (Ikeda-Garcia et al., 2007;Koutinas et al., 2001;Manna et al., 2008;Miró et al., 2009). In addition, drug resistance to antimonials (Carrió and Portús, 2002) or allopurinol (Yasur-Landau et al., 2017) has been described in dogs. ...
Leishmaniosis due to Leishmania infantum is a complex infection that can affect both humans and dogs, and present a wide range of clinical signs and clinicopathological abnormalities. The conventional treatment of this disease is challenging due to the fact that complete parasitological cure commonly does not occur. Furthermore, treatment of the disease with the conventionally used drugs has several shortcomings. These include the need for long-term treatment, side effects and the formation of drug resistance. Moreover, it is important to highlight that the host immune responses play a crucial role in the outcome of this infection. For this reason, the use of immunotherapy in clinical leishmaniosis to improve the result of treatment with the conventional anti-leishmanial drugs by enhancing the immune response is imperative. The aim of this review is to provide a comparative overview of the wide range of immunotherapeutical approaches and strategies for the treatment of L. infantum infection in animals focusing on dogs.
... Carbohydrate-based natural products constitute a very potent group of compounds with promising prospects as future drugs [1]. Currently, there are many carbohydrate natural product derived compounds that are already drugs and these include: The polysaccharides, tragacanth (diarrhea and constipation) [2], astragalus (cancer) [3], lentinan (cancer and hepatitis B) [4], tremella (cancer and chronic bronchitis) [5,6], icodextrin (peritoneal dialysis) [7] and pentosan polysulfate (interstitial cystitis) [8]; the oligosaccharides, lactulose (constipation and hepatic encephalopathy) [9], sucralfate (active duodenal ulcers) [10]; and the monosaccharides, miglitol (anti-diabetic) [11] and meglumine (excipient) [12]. ...
The Mycobacterium sp. BRS2A-AR2 is an endophyte of the mangrove plant Rhizophora racemosa G. Mey., which grows along the banks of the River Butre, in the Western Region of Ghana. Chemical profiling using 1H-NMR and HRESI-LC-MS of fermentation extracts produced by the strain led to the isolation of the new compound, α-d-Glucopyranosyl-(1→2)-[6-O-(l-tryptophanyl)-β-d–fructofuranoside] or simply tortomycoglycoside (1). Compound 1 is an aminoglycoside consisting of a tryptophan moiety esterified to a disaccharide made up of β-d-fructofuranose and α-d-glucopyranose sugars. The full structure of 1 was determined using UV, IR, 1D, 2D-NMR and HRESI-LC-MS data. When tested against Trypanosoma brucei subsp. brucei, the parasite responsible for Human African Trypanosomiasis in sub-Saharan Africa, 1 (IC50 11.25 µM) was just as effective as Coptis japonica (Thunb.) Makino. (IC50 8.20 µM). The extract of Coptis japonica (Thunb.) Makino. is routinely used as laboratory standard due to its powerful antitrypanosomal activity. It is possible that, compound 1 interferes with the normal uptake and metabolism of tryptophan in the T. brucei subsp. brucei parasite.
... As outlined above, numerous quantitative and qualitative real-time PCR-based assays to be used in veterinary or human medicine have been published recently, targeting different genetic markers and for application to different types of samples [51,[59][60][61][62][63][64][65][66][67][68][69]. The main features of qPCR assays targeting kDNA, rDNA or other DNA regions are summarized in Table 1, Table 2 and Table 3, respectively. ...
Leishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field.
... 50% of the patients still experienced at the end of M2 values above the reference limits which can be obtained only six months after the treatment ( Sasanelli et al. 2007) so the observed decreases demonstrate a good response to treatment and can interpreted as a significant reduction of the parasite in the patients resulting in an improvement of health ( Sarro et al. 2010). Clinical cure is often achieved with the used therapy ( Noli & Auxilia 2007, Paradis et al. 2012), nevertheless the patients still may continue to carrier the Leishmania (Geisweid, Mueller, Sauter-Louis, Hartmann 2012, Pennis et al. 2005, Ikeda-Garcia et al. 2007, Manna et al. 2008). , Ikeda-Garcia 2007), it was possible to notice differences between the FRG and SRG regarding to variations on the PT, β-globulin, and γglobulin fractions of proteinogram. ...
... Evidence for a relationship between parasitic load and clinical manifestations has been previously reported in dogs with leishmaniasis [25]. However, a lack of correlation between changes in clinical signs and parasitic load has also been reported [26][27][28], and it is known that anti-leishmanial treatments do not eliminate all parasites [27,[29][30][31][32][33][34][35]. Serological CanL titers were not repeated after treatment because, although they have been recently reported as potentially useful shortly after treatment [36], data in the literature support that a minimum period of 6 months is required to find significant changes in IFI results [5]. ...
Treatment of canine leishmaniasis (CanL) represents a challenge. Due to the high prevalence of renal disease associated to CanL, it is important to find an effective drug that does not damage the kidneys. Marbofloxacin has been shown to be effective and well tolerated in non-azotemic dogs with leishmaniasis. To evaluate the safety and efficacy of marbofloxacin in dogs with leishmaniasis and decreased renal function, 28 dogs suffering from leishmaniasis and chronic kidney disease (CKD) were treated with oral marbofloxacin at 2 mg/Kg/day for 28 days. During treatment dogs were assessed by performing weekly physical exams, measuring blood pressure and evaluating blood and urine parameters. Lymph node aspirations were also obtained at days 0 and 28. The global clinical score decreased significantly, from 6.2±3.4 to 4.7±3.1 (p = 0.0001), after treatment. Marbofloxacin also decreased parasitic load in 72% of the dogs. No significant differences in plasma creatinine, urine specific gravity, urinary concentrations of cystatin C, ferritin and urinary protein loss were detected during treatment. A transient but significant decrease in blood pressure was detected up to day 14 (from 180.1±36.6 to 166.0±32.7 mmHg; p = 0.016). Moreover, dogs showed a significant increase in plasma albumin concentration (from 15.0±5.2 to 16.6±3.9 g/L; p = 0.014) and a significant decrease in globulin concentration (from 59.0±18.1 to 54.1±18.0 g/L; p = 0.005). The results demonstrate that, in addition to being effective for treatment of CanL, marbofloxacin is a very safe drug in dogs with CKD and leishmaniasis.
... Limitations of the serological and parasitological tests point to the need for developing and implementing more sensitive and specific techniques, such as molecular-based reactions (Grimaldi and Tesh, 1993;Miró et al., 2008;Reale et al., 1999;Solano-Gallego et al., 2001). Molecular techniques allow quantification of the parasite load when quantitative PCR (qPCR) is used (Francino et al., 2006;Manna et al., 2004), either during the course of the natural or experimental infection or after treatment (Maia and Campino, 2008;Manna et al., 2008;Martinez et al., 2011;Pennisi et al., 2005). ...
Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific tech- nique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same de- tection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the pres- ence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator’s hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.
... However, this drug was reported to be quite toxic to humans, expensive and inefficient to the various species of Leishmania [1]. Therapy with meglumine and allopurinol antimoniate promotes not only a clinical improvement but also a marked decrease in parasitic load on blood, skin, and lymph nodes [2]. The treatment usually lasts for more than 6 months in cases of large lesions and lesions located in the joints or face [3]. ...
The aim of this study is to evaluate the effects of ApPDT (antiparasitic photodynamic therapy) on the interaction of Leishmania braziliensis with J774 macrophages, used as a photosensitizer, methylene blue associated with red laser. The tests are in triplicate and the samples divided into four groups: control, photosensitizer, laser, and ApPDT. The photosensitizer used was the methylene blue at concentration of 12.5 μg/mL. The parameters of the laser were λ = 660 nm, 40 mW, and 8.4 J/cm². Samples are analyzed by optical microscopy through the identification and counting of infected and uninfected macrophages, parasite load, infectivity, and infection index. Statistical analysis used ANOVA test with Tukey post-test, being considered statistically significant p < 0.05. The analysis of the interaction tests shows that the infection rate in the ApPDT group in relation to the control group presents a statistically significant reduction (p < 0.0001) of 71% at both 24 and 48 h (p < 0.0001) of 62%. ApPDT reduces the number of macrophages infected by Leishmania braziliensis, as well as the number of intracellular parasites, being a possible alternative therapy in the treatment of cutaneous leishmaniosis.
... Researchers, clinical laboratories, and insurers choose laboratory test requirements to maximize the relationship between sensitivity, specificity, simplicity and cost-benefits (22)(23)(24)(25). Molecular techniques could play an important role in detection and the monitoring of therapy in humans and animals leishmaniasis (26,27). Based on our study, the three molecular methods confirmed that all 50 positive sam-ples belong to L. tropica species. ...
Background
To compare three molecular methods, PCR-RFLP for internal transcribed spacer, PCR sequencing and high resolution melting analysis shown reliable sensitivity and specificity for detecting Leishmania tropica as a model for cutaneous leishmaniasis (CL) as the perspective overview for scientific and economic approaches.
Methods
This study was carried out between 2015 and 2016 in Leishmaniasis Research Center in Kerman University of Medical Sciences, Kerman, Iran. The positives smears (n=50) were obtained from patients referred from the health clinics in a major anthroponotic CL (ACL) focus, southeastern Iran. Only smear preparations with the same grade were selected according to the method described by the WHO for future PCR assays.
Results
All three molecular methods had capability to identify positive samples at species level with the same specificity and sensitivity. However, these techniques were different in simplicity, consuming time, and cost effectiveness. Although additional enzymatic process in PCR-RFLP provided good resolution to find Leishmania species but this would cause time and cost increases.
Conclusion
HRM (high resolution melting) is a relatively new technique that allows direct characterization of PCR amplicons in a closed system with more simplicity, cost effectiveness and time-consuming compared with other PCR-based assays for epidemiological or clinical identification purposes.
... Quantitative real-time PCR (qPCR) is a method that permits a quantitative analysis of the samples and the analysis of a large number of samples, resulting in a more accurate and sensitive diagnosis (Francino et al., 2006;Manna et al., 2008aManna et al., , 2008b. ...
Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300–350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis.
... Sie ermöglicht den DNA-Nachweis aus verschiedenen Flüssigkeiten und Bioptaten. Am sensitivsten sind Proben aus Knochenmark, Milz, Lymphknoten oder Haut (17,20,32). Als weiteres Diagnostikum steht die Untersuchung auf Antikörper gegen L. infantum zur Verfügung. ...
Zusammenfassung
Ein aus Spanien stammender, 3-jähriger Labrador-Retriever-Rüde wurde aufgrund einer seit mehreren Monaten bestehenden Lahmheit der linken Hintergliedmaße vorgestellt. Die orthopädische Untersuchung ergab eine Schmerzhaftigkeit bei Manipulation des linken Tarsus. Röntgenologisch und computertomographisch ließen sich polyostotische, aggressive osteolytische Knochenläsionen mit beginnender erosiver Arthritis nachweisen. Durch Untersuchung eines Knochenbioptats mittels PCR konnte über einen direkten Erregernachweis die Diagnose Leishmaniose gestellt werden. Drei Wochen nach Beginn einer Behandlung mit Allopurinol war der Hund lahmheitsfrei. Acht Monate nach Therapiebeginn zeigte sich röntgenologisch eine mittelgradige Regression der Osteolysen.
... A variety of clinical samples have been used for the detection of Leishmania DNA such as whole blood, buffy coat, bone marrow, lymph node, spleen, conjunctival swabs [14,15] and other biological samples such liver, lung, heart, penis, vagina, testis, semen, uterus, placenta, kidney, intestine, milk and urine [16] and more recently nasal, ear and oral swabs [17,18]. Bone marrow, lymph node, spleen and skin are the tissues presenting the highest sensitivity for the diagnosis of canine leishmaniasis [11,19]. The same holds true for the non invasive sampling techniques using conjunctival swabs [15,17]. ...
... Others found PCR being more sensitive (Wang et al., 2011, Chargui et al., 2009. PCR from tissue samples (e. g., skin, lymph nodes and bone marrow) are considered more sensitive than from blood samples (Manna et al., 2008, Maia et al., 2009, Strauss-Ayali et al., 2004. ...
Knowledge on the prevalence of the zoonotic and animal pathogens in dogs from Albania is thus far limited. Samples were collected from 119 police dogs which were deployed throughout Albania and which were living in close relationship with humans. Direct and/or indirect methods were used to examine 119 whole blood and 117 serum samples for the protozoans Toxoplasma gondii (IFAT) and Neospora caninum (IFAT), various vector-borne pathogens of zoonotic and veterinary concern (SFG rickettsiae [ELISA, IFAT], Anaplasma spp. [PCR, IFAT], Ehrlichia canis [PCR, IFAT], Babesia spp. [PCR, IFAT], Leishmania infantum [PCR, IFAT], Hepatozoon canis [PCR], Dirofilaria immitis [ELISA]), and haemotropic mycoplasmas [PCR]. DNA of L. infantum and A. platys was detected in the blood of four and two dogs, respectively, but all samples were negative for DNA of E. canis, Babesia spp., H. canis and haemotropic mycoplasmas. Dirofilaria immitis antigen was detected in 11.2% of the serum samples. The overall seroprevalences were: SFG rickettsiae (ELISA), 71.5%; Rickettsia conorii (IFAT), 61.5%; T. gondii, 65.0 %; Anaplasma spp., 31.6%; E. canis, 17.9%; L. infantum, 12.0%; N. caninum, 12.0% and Babesia canis, 3.4%. Seven dogs were PCR-negative and seronegative for all tested pathogens. Significantly more dogs from South Albania (24.3%) had antigen of D. immitis than dogs from North Albania (5.4%, p = 0.046). There was no difference in the geographical distribution of exposure to any other pathogen. Age was found to be a risk factor (p < 0.05) for exposure to T. gondii, SFG rickettsiae, Anaplasma spp. and L. infantum. Co-exposure to up to five vector-borne pathogens was found in 51.5% of the 103 dogs that were seropositive for at least one vector-borne pathogen. Results of this study indicate a considerable risk of infection for dogs and/or humans with T. gondii and N. caninum and a range of zoonotic vector-borne bacterial and protozoan pathogens.
... Different studies of leishmaniosis such as animal models, vectorial capacity, diagnosis, drug efficacy have been investigated using Real-time PCR (Talmi-Frank et al., 2010). The treatment of Leishmania infected dogs using meglumine antimoniate and allopurinol was monitored using quantitative PCR (Manna et al., 2008). A new version of real-time PCR i.e. multiplex real-time PCR was used for identification of Sarcocystis spp. in cattle in a single reaction (Moré et al., 2013). ...
The authors present an update of the molecular and serological techniques used in veterinary parasitology.
The application of molecular and serological techniques in veterinary parasitology is increasing worldwide. Several
new molecular techniques have been made available for sensitive and specific diagnosis of parasitic diseases. These tests
may be either nucleic acid or protein based assays. In nucleic acid based assays, PCR and its variants such as nested
PCR, multiplex PCR are the most frequently used tools. The parasitic nucleic acid target sequences are DNA and
ribosomal RNA. The DNA sequences provide high specificity for the identification of parasite in biological samples.
Real-Time PCR is also increasingly in use since its cost is decreasing. Some other tests such as RAPD, AFLP and
RFLP are also used for several parasitic disease diagnoses. Moreover now, complete genome sequencing of many of
parasites has been achieved. The complete genome data also provides suitable gene sequence which, when cloned and
expressed provides antigens for diagnosis and vaccine preparation. Similarly, protein based serological assays such
as ELISA, dot-ELISA, Fluorescence antibody tests have also been used for parasitic diagnosis. However, the use of
molecular tools in veterinary parasitology remains restricted to research rather than use in field as diagnostic tool.
Keywords | Parasites, Molecular diagnosis, PCR, RFLP, ELISA
... Monoclonal antibodies are useful for identification of species in cultured strains but are not as amenable to direct analysis of clinical specimens. Recently, conventional parasitological and serological techniques have been integrated by the more sensitive and specific polymerase chain reaction (PCR) assay (Grimaldi et al 1987, Leontides et al 2002, Manna et al 2008, Strauss-Ayali et al 2004, Schonian et al 2003). PCR has been shown to overcome problems such as the low sensitivity found with microscopic examination of tissue smears, and the limited predictive value of serology where the results may be affected by persistent antibodies or immunosuppression (Leontides et al 2002). ...
Leishmaniasis is caused by parasitic protozoa of the genus Leishmania. Cutaneous leishmaniasis (CL) is a complex disease with wide spectrum of clinical manifestations. In order to identify leishmania species causing CL in Isfahan by a definite molecular technique (PCR method), this study was undertaken over 2010-2011. 124 Patients with suspicious lesion of Leishmaniasis and positive direct smear from lesion were selected. Samples were cultured in NNN and RPMI 1640 media Negative and positive control and clinical samples was applied for PCR in the same condition. In the next step, standard PCR was carried out using classic protocol. From 124 patients, 111 (89.51%) cases were infected as L. major and 12 (9.67%) cases were infected by L. tropica, However only in one patient simultaneous infectious with both L. major and L. tropica was identified by PCR techniques which could not be possible in microscopy. L. major was the most prevalent species in the studied patients (p-value<0.001).
... Monoclonal antibodies are useful for identification of species in cultured strains but are not as amenable to direct analysis of clinical specimens. Recently, conventional parasitological and serological techniques have been integrated by the more sensitive and specific polymerase chain reaction (PCR) assay (Grimaldi et al 1987, Leontides et al 2002, Manna et al 2008, Strauss-Ayali et al 2004, Schonian et al 2003). PCR has been shown to overcome problems such as the low sensitivity found with microscopic examination of tissue smears, and the limited predictive value of serology where the results may be affected by persistent antibodies or immunosuppression (Leontides et al 2002). ...
Leishmaniasis is caused by parasitic protozoa of the genus Leishmania. Cutaneous leishmaniasis (CL) is a complex disease with wide spectrum of clinical manifestations. In order to identify leishmania species causing CL in Isfahan by a definite molecular technique (PCR method), this study was undertaken over 2010-2011. 124 Patients with suspicious lesion of Leishmaniasis and positive direct smear from lesion were selected. Samples were cultured in NNN and RPMI 1640 media Negative and positive control and clinical samples was applied for PCR in the same condition. In the next step, standard PCR was carried out using classic protocol. From 124 patients, 111 (89.51%) cases were infected as L. major and 12 (9.67%) cases were infected by L. tropica, However only in one patient simultaneous infectious with both L. major and L. tropica was identified by PCR techniques which could not be possible in microscopy. L.major was the most prevalent species in the studied patients (p-value<0.001).
Canine leishmaniosis (CanL) is a growing health problem for which vaccination is a crucial tool for the control of disease. The successful development of an effective vaccine against this disease relies on eliciting a robust and enduring T-cell immune response involving the activation of CD4+ Th1 and CD8+ T-cells. This study aimed to evaluate the immunogenicity and prophylactic efficacy of a novel nanovaccine comprising a multi-epitope peptide, known as HisDTC, encapsulated in PLGA nanoparticles against Leishmania infantum infection in the murine model. The encapsulation strategy was designed to enhance antigen loading and sustain release, ensuring prolonged exposure to the immune system. Our results showed that mice immunized with PLGA-encapsulated HisDTC exhibited a significant reduction in the parasite load in the liver and spleen over both short and long-term duration. This reduction was associated with a cellular immune profile marked by elevated levels of pro-inflammatory cytokines, such as IFN-γ, and the generation of memory T cells. In conclusion, the current study establishes that PLGA-encapsulated HisDTC can promote effective and long-lasting T-cell responses against L. infantum in the murine model. These findings underscore the potential utility of multi-epitope vaccines, in conjunction with appropriate delivery systems, as an alternative strategy for CanL control.
Leishmaniasis is a widespread but still underdiagnosed parasitic disease that affects both humans and animals. There are at least 20 pathogenic species of Leishmania , most of them being zoonotic. The diagnosis of leishmaniasis remains a major challenge, with an important role being played by the species of parasites involved, the genetic background, the immunocompetence of the host. This paper brings to the fore the sensitivity of the balance in canine and human leishmaniasis and addresses the importance of the host’s immune response in establishing a correct diagnosis, especially in certain cases of asymptomatic leishmaniasis, or in the situation the host is immunosuppressed or acquired leishmaniasis through vertical transmission. The methods considered as a reference in the diagnosis of leishmaniasis no longer present certainty, the diagnosis being influenced mostly by the immune response of the host, which differs according to the presence of other associated diseases or even according to the breed in dogs. Consequently, the diagnosis and surveillance of leishmaniasis cases remains an open topic, requiring new diagnostic methods adapted to the immunological state of the host.
Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum transmitted by insects of the genus Phlebotomus and Lutzomyia in the Old and New Worlds, respectively. It is a severe, systemic and potentially fatal disease. Despite the availability of some prophylactic and therapeutic tools, their effectiveness is questionable. Although neutrophils (PMN) are the first line of defense of the organism, rapidly phagocytizing the parasite after inoculation, macrophages (MØ) are the definitive host cells, supporting their multiplication and spread. This study aimed to identify the effector mechanisms activated by PMN in response to L. infantum promastigotes and the impact of PMN-MØ interaction in the initial phase of canine infection by determining its contribution to infection control or, on the contrary, the establishment of clinical disease. Clinically and analytically healthy animals who tested negative for the main infectious and parasitic diseases were selected and PMN-Leishmania cultures were established. It was found by flow cytometry and microscopic observation that L. infantum promastigotes are efficiently phagocytized by PMN in the majority of the dogs, activating oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) mechanisms, but preventing the release of neutrophil extracellular traps (NET). Furthermore, promastigotes and culture supernatants induced PMN migration, but the prior contact with Leishmania inhibited chemotaxis, which contributes to PMN retention at the inoculation site. The interaction with the parasite had a negative impact on PMN viability, promoting secondary necrosis. PMN-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survived and maintained their proliferative capacity, contributing to the establishment of the infection. MØ differentiated from blood monocytes underwent major morphological changes in order to contain the parasite and also released Histone H1, but its leishmanicidal effect was not yet proven in L. infantum promastigotes. Parasite transfer to MØ was confirmed by flow cytometry in co-cultures of MØ-infected PMN whose extracellular parasites were removed. The microscopic observation of these co-cultures showed that infected PMN
efferocytosis and probably phagocytosis of parasites released from apoptotic PMN are crucial for parasite transfer to the definitive host cell. Unexpectedly, PMN-MØ
interaction activated nitric oxide production and induced NET release, which can
contribute to parasite containment and to the early control of the infection. These findings broaden the knowledge of L. infantum infection in the dog and can shed light in the design of new therapeutic and prophylactic tools, leading to a marked reduction in CanL.
Canine infections with Leishmania (L.) infantum are gaining significance in Germany due to rising numbers of dogs imported from endemic countries, frequent travel and changing of climatic conditions in Central Europe. Dogs without any clinical signs suspicious for vector-borne infections imported from other countries to Germany should be tested immediately after import and 6 months later. In dogs with clinical signs suspicious for leishmaniosis, direct and indirect detection methods of the
pathogen as well as hematology, biochemistry, serum protein electrophoresis and C-reactive protein are recommended. For treatment and monitoring of canine leishmaniosis, the LeishVet-guidelines are highly recommended. Different therapeutic options include first-line, second-line, and third-line drugs. For dose adjustments of allopurinol, the “step plan” should be taken in consideration. Due to climatic changes, habitats of sandflies as transmitting vectors of leishmaniosis are expanding. t to vectorial transmission mating, transplacental infections, bite wounds, and blood transfusions were described in canine leishmaniosis. Additionally, L. infantum is a zoonotic vector-borne infectious pathogen, which is important regarding the “One-health”-aspect.
La leishmaniosis visceral (LV) es una de las enfermedades parasitarias mas importantes a nivel mundial. En zonas endemicas de leishmaniosis visceral zoonotica (LVZ), los perros son considerados el reservorio mas importante y las altas prevalencias de leishmaniosis canina (LCan) se encuentran asociadas con la transmision de la infeccion a humanos. Ante la emergencia de LV humana en la ciudad de Posadas en 2006, se planteo un primer estudio a fin de determinar la implicacion de los perros como reservorio de LVZ en este contexto; asi como, en caso afirmativo, caracterizar molecularmente la especie o especies encontradas en los mismos. La infeccion por Leishmania fue confirmada por metodos parasitologicos directos y/o indirectos en 63 (57,3%) de los 110 perros estudiados y se identifico a L. infantum como unica especie implicada. Posteriormente, se diseno un segundo estudio, con el objetivo de determinar la prevalencia e incidencia acumulada (IA) de la infeccion por L. infantum, en los perros domesticos de la ciudad de Posadas, e investigar posibles factores de riesgo asociados a la infeccion por L. infantum en estos canes. Para estudiar la prevalencia de la infeccion se llevo a cabo un muestreo aleatorio simple (MAS), y esta resulto del 22,3% (78/349) (17,6%-27,8%). La georreferenciacion de los perros examinados, permitio observar un patron de distribucion homogeneo de los perros infectados en la ciudad. Los canes asintomaticos infectados representaron en este estudio el 55,1% (43/78) del total de infectados, pudiendo jugar un papel importante en la transmision natural de la infeccion. Se observo un mayor riesgo en la adquisicion de la infeccion por Leishmania en los perros que duermen fuera de la casa. La IA en los perros que resultaron negativos a la infeccion por Leishmania al ano del MAS resulto del 10,5% (4,7%-16,3%), y mostro el mismo patron de distribucion espacial. Se observa que en el estudio de IA el porcentaje de perros asintomaticos positivos 3/13 (23,1%), es mucho mas bajo que el observado en el estudio de prevalencia (43/78, 55,1%). Los resultados de los metodos parasitologicos directos e indirectos empleados para determinar la infeccion por L. infantum en la poblacion canina de la ciudad de Posadas, resultaron concordantes; y se observo que la sintomatologia relacionada con LCan en los perros domesticos es inespecifica, ya que el 75,9% (110/145) de los perros sintomaticos no se encontraban infectados. Por ultimo, se compararon los resultados de los metodos parasitologicos directos e indirectos en dos poblaciones caninas diferentes, para determinar el grado de asociacion entre las variables estudiadas y la infeccion por Leishmania en un ejemplo de poblacion dispersa (MAS en perros domesticos de la ciudad) vs poblacion hacinada (perros de la protectora de animales ?El Refugio). Asi como describir en ambos contextos epidemiologicos la distribucion de los canes expuestos e infectados en relacion a los signos clinicos presentados y a los resultados de las pruebas diagnosticas. Ademas, se analizo la variabilidad genetica de los minicirculos del kDNA de L. infantum en los perros parasitologicamente positivos de ambas poblaciones. Los resultados mostraron que las poblaciones hacinadas poseen mayores prevalencias de infeccion por Leishmania que las dispersas (45,7% vs 22,3%). Y, por tanto, no serian apropiadas para estimar la prevalencia/incidencia real de la infeccion en la ciudad. La variabilidad genetica de L. infantum en los perros de la ciudad de Posadas resulto ser escasa, identificandose unicamente dos genotipos. En vista de los resultados la LVZ en la ciudad de Posadas se encuentra bien establecida y podria tratarse de un foco de reciente introduccion. El conocimiento aportado a la epidemiologia de la infeccion por L. infantum en los canes de la ciudad de Posadas, debe contribuir al establecimiento de medidas de prevencion y control de la enfermedad tanto en canes como en humanos
Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.
This case describes a dog with severe epistaxis due to an infection with Leishmania infantum . The dog was hypovolemic and anaemic at presentation. Standard treatment with sedation and application of gauzes with epinephrine was unsuccessful. The bleeding was temporarily controlled by applying two balloon catheters in the right nasal passage under general anaesthesia. The bleeding recurred after one of the catheters got displaced. Ultimately, the right common carotid artery was ligated, after which right-sided epistaxis did not recur.
Re-emergence and geographic expansion of leishmaniasis is accelerating efforts to develop a safe and effective Leshmania vaccine. Vaccines using Leishmania recombinant antigens, such as LiHyp1, which is mostly present in the amastigote parasite form, are being developed as a next generation to crude killed parasite-based vaccines. The main objective of this work was to develop a LiHyp1-based vaccine and determine if it can induce protective immunity in BALB/c mice when administered using a dissolvable microneedle (DMN) patch by the skin route. The LiHyp1 antigen was incorporated into cationic liposomes (CL), with or without the TLR9 agonist, CpG. The LiHyp1-liposomal vaccines were characterized with respect to size, protein encapsulation rates and retention of their physical characteristics after incorporation into the DMN patch. DMN mechanical strength and skin penetration ability were tested. A vaccine composed of LiHyp1, CpG and liposomes and subcutaneously injected or a vaccine containing antigen and CpG in DMN patches, without liposomes, induced high antibody responses and significant levels of protection against L. donovani parasite infection. This study progresses the development of an efficacious leishmania vaccine by detailing promising vaccine formulations and skin delivery technologies and it addresses protective efficacy of a liposome-based dissolvable microneedle patch vaccine system.
The aim of this 6-month, randomized, blinded, controlled clinical trial was to compare the efficacy and safety of aminosidine-allopurinol combination with that of meglumine antimoniate-allopurinol combination for the treatment of leishmaniosis in dogs without stage III or IV chronic kidney disease. Forty client-owned dogs were randomly assigned to group A [n = 20; aminosidine (15 mg/kg, subcutaneously, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)] or group B [(n = 20; meglumine antimoniate (100 mg/kg SC, once daily, for 28 days) and allopurinol (10 mg/kg, per os, twice daily, for 6 months)]. Clinical and clinicopathological evaluations, parasitic load measurement (lymph node and bone marrow microscopy, bone marrow real-time PCR), specific serology and leishmanin skin test (LST) were performed at baseline (time 1) and after 14 (time 2), 28 (time 3), 60 (time 4) and 180 (time 5) days. Both treatments were safe and resulted in significant clinical and clinicopathological improvement, reduction of parasitic load and of indirect immunofluorescence antibody test (IFAT) titer and induction of positive LST. There was no significant difference between groups with regards to the primary outcome measures of the trial that included the proportion of dogs that presented severe treatment-related side effects, were cured and were parasitologically negative at time 5. However, some (proportion of dogs that presented no clinical signs, no hyperglobulinemia and negative serology at time 5) secondary outcome measures showed significant differences in favor of the meglumine antimoniate-allopurinol treatment arm. Treatment-related death occurred in one dog in each group, while injection site reactions appeared at a similar frequency in both groups. Due to the differences in some secondary outcome measures in association with the low power of this trial, it cannot be definitively concluded that the two treatments are equally effective. Therefore, the aminisodine-allopurinol combination cannot be proposed as a first-line treatment of CanL but rather as a second-line treatment that may be particularly useful to avoid repeated administration of meglumine antimoniate and in countries where the latter is not available or registered.
Guidelines on "Diagnosis, Classification, Therapy, Monitoring and Prevention of Canine Leishmaniasis" are formulated by the Canine Leishmaniasis Working Group (CLING) with the aim to offer to the veterinarian the correct overview on pathophysiology, diagnosis, therapy, and prevention of canine disease caused by Leishmania spp. CLING activity grounds on evidence based medicine; in case of controversies or absence of evidence in veterinary literature, a consensus is reached among the members. These guidelines are intended to veterinarians and are not to be thought as totally exhaustive or unchangeable. The aim of this paper is to provide a tool to use in most conditions but that cannot be able to work out all the issues related to canine leishmaniasis. Guidelines are updated annually based on veterinary literature. Guidelines and their updates are published on "Veterinaria" journal and edited on www.gruppoleishmania.org web-site after their approval by the CLING members. Dogs affected by leishmaniasis are classified in 5 stages: 1) Stage A - exposed; 2) Stage B - infected; 3) Stage C - sick; 4) Stage D - sick with severe clinical signs; 5) Stage E - a: not responsive to therapy; E - b: early relapse. After revision of literature, the consensus was reached for the following therapeutic approach to canine leishmaniasis: 1. Meglumine Antimoniate - Allopurinol combination is the first choice in stage 8, C and D dogs. Meglumine Antimoniate is administered subcutaneously for 4 weeks at, the daily dosage of 100 mg/kg, Allopurinol at the dosage of 10 mg/kg q12h po for at least 4-6 months. Meglumine Antimoniate administration can be divided twice daily (50 mg/kg q12h) and continued up to 8 weeks. 2. An alternative therapeutic approach could be considered in Stage E dogs, using other drugs, when other diseases and concurrent factors (inadequate drug protocol, absence of owner compliance) have been ruled out. 3. Allopurinol administered as single drug at the dosage of 10-20 mg/kg q12h po for at least 3-6 months can be considered an alternative therapeutic approach in all patients presenting contraindications for Meglumine Antimoniate or showing other anti-leishmania drugs side effects. Also, Allopurinol could be suggested when owner compliance is inadequate.
Canine leishmaniasis is a worldwide disease caused by Leishmania infantum. Transmission takes place through sand flies which predominantly occur in subtropical to tropical regions especially important for Europe in the Mediterranean area. The reaction of the body's immune system facing a leishmania infection is crucial for the development of a clinical disease or chronic infection. Asymptomatic disease takes place in face of an intact cellular immune system while diminished cellular immunity coincides with clinical symptoms. As leishmaniasis is a systemic disease, a wide variety of clinical symptoms may be encountered. Renal disease with glomerulonephrititis due to immune complex depositions is the most common cause of death in patients with leishmaniasis. Detection of leishmania-specific antibodies is the best screening method. Equivocal results can be underlined by PCR or cytological examinations. Therapy of leishmaniasis is tedious and recurrence is common. N-methylglucamine antimonate (Glucantime ®, Merial GmbH, D) single or in combination with allopurinol is one of the best therapeutic strategies, although gastrointestinal disease and nephropathologies may be observed as side effects. Decreased glomerular filtration rate will increase half-life of allopurinol. Therefore control of kidney parameters is warranted in every patient with leishmaniasis. Third drug of choice is miltefosin (Milteforan ®, Virbac Animal Health) alone or in combination with allopurinol which has similar side effects as n-methylglucamine antimonate. Disease prevention is most important in limiting spread of leishmania organism worldwide. Thereby especially collars and spot-on preparations as well as the new vaccine CaniLeish (Virbac Animal Health) are effective.
Leishmania species cause a variety of in part fatal diseases in man and animals, the prominent veterinary manifestation being canine leishmaniasis caused by L. infantum (syn. L. chagasi), which also causes visceral leishmaniasis in humans. Canine leishmaniasis is common in the Mediterranean basin, Central and South America, the Middle East, and China, and the infected dogs, depending on the spread of transmitting vectors, represent a permanent threat to human health as reservoir of the parasite. The status of leishmaniasis treatment, as well as recent progress, is reviewed with special emphasis on canine leishmaniasis, and the problems arising from L. infantum being both a zoonotic and anthroponotic parasite. In essence, treatment of canine leishmaniasis relies on the same drugs used for human leishmaniasis (i.e., pentavalent antimonials, amphotericin, paromomycin and miltefosine with or without drug delivery systems). Despite demonstrated clinical efficacy, none of these drugs reliably achieves a parasitological cure in dogs, which implies the risk of relapses, spread of the disease within the dog population, human infection, and development of drug resistance. A critical evaluation of the present therapeutic scenario reveals that more efficacious, safer, and affordable drugs are required to solve the veterinary and public health problems resulting from the infections by L. infantum and Leishmania species in general.
A polymerase chain reaction (PCR) technique for the diagnosis of canine leishmaniasis on bone marrow samples was developed which amplified a 120 bp DNA fragment of the Leishmania kinetoplast DNA, common to all Leishmania species. Forty-five of 46 dogs in which leishmaniasis had been diagnosed were positive with the PCR technique, whereas none of 41 healthy dogs gave a positive result. Fifteen dogs with leishmaniasis that had been treated for six months with N-methylglucamine antimoniate and allopurinol were also investigated. Seven were positive, implying that they remained infected despite the resolution of their clinical signs.
We studied and compared the prevalence of Leishmaniainfection and the seroprevalence and the prevalence of canine leishmaniasis in an area where canine leishmaniasis is endemic.
One hundred dogs living on the island of Mallorca (Spain) were studied. In this study, we clinically examined each dog for
the presence of symptoms compatible with leishmaniasis, determined the titer of anti-Leishmania antibodies, and investigated the presence of Leishmania DNA by PCR in skin, conjunctiva, and bone marrow samples of each dog. The prevalence of the disease and the seroprevalence
were 13 and 26%, respectively. In 63% of the dogs,Leishmania DNA could be detected by PCR in at least one of the tissues studied. The results of positive PCR in the bone marrow, the
conjunctiva, and the skin were 17.8, 32, and 51%, respectively. The prevalence of the infection, 67%, was calculated using
all animals that were seropositive and/or positive by PCR with any tissue. The results showed that the majority of dogs living
in an area where canine leishmaniasis is endemic are infected by Leishmania and that the prevalence of infection is much greater than the prevalence of overt Leishmania-related disease.
The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.
The objectives of this study were to compare the sensitivities and reliabilities of different PCR methods for the diagnosis and epidemiological study of canine visceral leishmaniasis (CVL) using dog blood. We chose to work with peripheral blood, as this type of sampling is noninvasive, straightforward, and easy to repeat. Six PCR methods were compared: three primer pairs target genomic DNA, and the other three target kinetoplast (mitochondrial) DNA. Sensitivity, specificity, reproducibility, and ease of interpretation without hybridization were evaluated for each method. The assessment was first performed using artificial samples. All methods could detect less than one parasite per reaction tube. However, the sensitivities varied among the different methods by a factor of 500 on purified cultivated parasites and by a factor of 10,000 on seeded dog blood samples (i.e., from 10 to 10(-3) parasite per ml of blood for the latter). Only four methods were found sufficiently reliable for the diagnosis of CVL. They were tested on 37 dogs living in an area of endemicity and grouped according to clinical status and specific serology. Only the two methods targeting kinetoplast DNA (K13A-K13B and RV1-RV2) could detect the parasite in 100% of symptomatic infected dogs. Similarly, all seropositive dogs were found PCR positive by these methods versus 62% by the genomic-DNA-based methods. Finally, these kinetoplast-based methods proved clearly superior to the others in the detection of Leishmania in asymptomatic dogs. Our data allow the discussion of the advantages and drawbacks of highly sensitive versus moderately sensitive PCR methods in diagnosis and prevalence studies of CVL.
Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However,
monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly
accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying
a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of
L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity
over an at least 6-log DNA concentration range, corresponding to 0.1 to 104 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same
magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional
PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and
will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher
the unique properties of the life cycle of Leishmania spp .
Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation
of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of ≥95%
at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral
leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow,
skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with
fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0°C, respectively). All three groups could
be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient
of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed
higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR
is utilized for therapy monitoring.
Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides.
A real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to reach a sensitivity of 0.0125 parasites/ml of blood. In order to analyze the incidence of heterogeneity and number of minicircles, we performed comparative PCR by using the Leishmania DNA polymerase gene as a reporter. Assays performed in both promastigote and amastigote stages showed variations among different L. infantum and Leishmania donovani strains and the stability of the minicircle numbers for a particular strain. Analysis of blood samples from a patient who presented with Mediterranean visceral leishmaniasis confirmed the reliability of such an assay for Leishmania quantification in biological samples and allowed an estimation of positivity thresholds of classical tests used for direct diagnosis of the disease; positivity thresholds were in the range of 18 to 42, 0.7 to 42, and 0.12 to 22.5 parasites/ml for microscopic examination, culture, and conventional PCR, respectively. At the time of diagnosis, parasitemia could vary by a wide range (32 to 188,700 parasites/ml, with a median of 837 parasites/ml), while in bone marrow, parasite load was more than 100 parasites per 10(6) nucleated human cells. After successful therapy, parasitemia levels remain lower than 1 parasite/ml. In the immunocompromised host, relapses correlate with an increase in the level of parasitemia, sometimes scanty, justifying the need for assays with high sensitivity. Such sensitivity allows the detection of Leishmania DNA in the blood of 21% of patients with no history of leishmaniasis living in the Marseilles area, where leishmaniasis is endemic. This technique may be useful for epidemiologic and diagnostic purposes, especially for the quantification of parasitemia at low levels during posttherapy follow-up.
Most of the experimental studies of Leishmania spp. infection require the determination of the parasite load in different tissues. Quantification of parasites by microscopy is not very sensitive and is time consuming, whereas culture microtitrations remain laborious and can be jeopardized by microbial contamination. The aim of this study was to quantify Leishmania infantum parasites by real-time polymerase chain reaction (PCR) using specific DNA TaqMan probes and to compare the efficacy of detection of this technique with a PCR-enzyme-linked immunosorbent assay (ELISA). For this purpose, spleen and liver samples from L. infantum-infected mice were collected during a 3-mo longitudinal study and analyzed by both methods. PCR-ELISA failed to quantify Leishmania spp. DNA in samples with very low or very high numbers of parasites. Real-time PCR was more sensitive than PCR-ELISA, detecting down to a single parasite, and enabled the parasite quantification over a wide, 5-log range. In summary, this study developed a method for absolute quantification of L. infantum parasites in infected organs using real-time TaqMan PCR.
Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify
the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the
effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative
nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA),
which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes
and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection
limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites
per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test
evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA
is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of
CL patients.
Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis.In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use.The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis.The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre ≥200), and 19% by microscopic examination of lymph node aspirates.
The aim of the present study was to evaluate the long-term clinical outcome for dogs with leishmaniasis that were treated with 3 different protocols: combined treatment with antimony and allopurinol, antimony alone, or allopurinol alone. Ninety-six dogs included in this study were determined to have leishmaniasis on the basis of (1) clinical features, (2) identification of the parasite in smears of lymph node, bone marrow aspirates, or skin biopsies, and (3) specific immunofluorescent assay. Three groups of dogs were defined: 45 dogs (group 1) were treated with antimony (100 mg/kg s.c. q24h) given concurrently for 1 month with allopurinol (15 mg/kg p.o. q12h), and then allopurinol alone for 8 months at the same dosage; 40 dogs (group 2) were treated with antimony alone according to the manufacturer's instructions (200 mg/kg s.c. q24h at 2-day intervals for 3-6 months); and 11 dogs (group 3) were treated with allopurinol alone (15 mg/kg p.o. q12h for 1-20 months). Information concerning signalment, history, physical examination findings, serologic testing and number of dogs becoming seronegative, outcome for each treated dog (clinical cure versus failure), and long-term survival were recorded. The numbers of the clinical cures versus failures were significantly different among the 3 groups (chi2 = 17.77, P < .001), between groups 1 and 2 (chi2 = 8.02, P < .01), between groups 2 and 3 (chi2 = 11.00, P < .01), and between groups 1 and 3 (chi2 = 16.52, P < .001). No significant difference between groups 1 and 2 was noted in the type of failure (relapse or death), serologic test results, and number of survival years (chi2 = 2.79, P > .05). The results of the present study indicate that antimony in combination with allopurinol produces better results than antimony alone or allopurinol alone for the treatment of the canine leishmaniasis. With combination treatment, duration of treatment with antimony is shorter and long-term administration of allopurinol is well tolerated.
Visceral leishmaniosis is a widespread and potentially fatal disease of dogs and humans common in the Mediterranean region, the Middle East, and South America. Canine leishmaniosis is most frequently treated with the drugs meglumine antimoniate, allopurinol, amphotericin B, or a combination of meglumine antimoniate and allopurinol. Therapy with the currently used drugs often achieves temporary clinical improvement and changes in immunologic parameters with restoration of the ability to mount parasite-specific cell mediated responses and decrease in anti-leishmanial antibody titers. However, treatment usually does not prevent relapse of disease or eliminate parasite carriage. Due to the current lack of an ultimate and effective therapy for canine leishmaniosis, new drugs, delivery systems and treatment strategies are necessary to achieve a consistent parasitological cure in infected dogs.
A total of 73 clinically healthy hunting dogs, experiencing an outdoor lifestyle and originating from an area where canine leishmaniasis is endemic, were included in the study. Polymerase chain reaction (PCR) and indirect immunofluorescent antibody test (IFAT) for Leishmania spp. were done on bone marrow and serum samples, respectively, obtained from all 73 dogs, just before the beginning of the sandfly season. PCR was found positive in 46/73 (63%) whereas, IFAT only in 9/73 (12.3%) of the dogs. The prevalence and the incidence of Leishmania infection by PCR were 61.9 and 47.1%, respectively. No association was found between the breed, age, sex, length of hair coat of the dog, urban or rural life and the presence of ample vegetation and water collections in the proximity of their living quarters, and the result of PCR. These findings clearly demonstrate that most of the dogs residing areas where leishmaniasis is endemic become infected but usually remain seronegative. Serological screening of the general canine population in these areas may result in an underestimation of the true prevalence of the infection rate.
Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis. In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use. The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis. The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre > or =200), and 19% by microscopic examination of lymph node aspirates.
A polymerase chain reaction (PCR) procedure using noninvasively obtained samples, for the identification of Leishmania infantum in canine tissues, was evaluated and compared with serologic testing and culture. A total of 92% of naturally infected, symptomatic,
seropositive dogs were found to be positive by use of DNA from conjunctival swabs. Spleen or lymph node aspirates were found
to be positive by PCR in 86% and by culture in 74% of these dogs. The sensitivity and specificity of conjunctival PCR were
92% and 100%, respectively. Experimentally infected dogs were found to be positive by conjunctival PCR already at 45 days
of infection (83%) and before seroconversion. PCR using noninvasively obtained conjunctival samples will be useful for epidemiological
studies and for direct diagnosis of canine visceral leishmaniasis.
In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.
The sensitivity and specificity of lymph node and bone marrow smear microscopy for the diagnosis of Leishmania infantum-infected dogs was evaluated in 79 dogs with leishmaniasis (Group A), 52 asymptomatically infected dogs (Group B), and 44 healthy noninfected dogs (Group C). Light microscopy examination included 10 to 1,000 oil immersion fields, and the density of Leishmania amastigotes was scored by a 0 to +6 scale. Using polymerase chain reaction as the gold standard, the specificity of lymph node and bone marrow cytology was 100%, whereas sensitivity ranged from 7.8% to 92.6%, being significantly higher in Group A compared with Group B. The amastigote scores were also significantly higher in Group A compared with Group B. These results indicate that lymph node and bone marrow cytology is a highly sensitive and specific method for the diagnosis of canine patent leishmaniasis, whereas its sensitivity is relatively low in asymptomatic infections.
Leishmaniases are a complex of world-wide diseases with a range of clinical and epidemiological features caused by Leishmania spp. of protozoan parasites. Among 15 well-recognised Leishmania species known to infect humans, 13 have zoonotic nature, which include agents of visceral, cutaneous and mucocutaneous forms of the disease in both the Old and New Worlds. Currently, leishmaniases show a wider geographic distribution and increased global incidence of human disease than previously known. Environmental, demographic and human behavioural factors contribute to the changing landscape of leishmaniasis, which includes increasing risk factors for zoonotic cutaneous leishmaniases and new scenarios associated with the zoonotic visceral leishmaniases. The latter consist of the northward spread of Leishmania infantum transmission in Europe and America, the identification of unusual mammal hosts, and the decline of HIV-Leishmania co-infections in southern Europe following the introduction of the highly active antiretroviral therapy. Few advances have been made in the surveillance and control of the zoonotic leishmaniasis, however a number of tools have been developed for the control of the canine reservoir of L. infantum. These include: (i) several canine vaccine candidates, in particular an FML Leishmania enriched fraction showing good clinical protection, has been registered in Brazil for veterinary use; (ii) a number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites. Laboratory and field studies have shown improved efficacy of these preparations for both individual and mass protection.
The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.
The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.
- L Manna
- S Reale
- E Viola
- F Vitale
- V Foglia Manzillo
- L M Pavone
- S Caracappa
- A E Gravino
Manna, L., Reale, S., Viola, E., Vitale, F., Foglia Manzillo, V., Pavone,
L.M., Caracappa, S., Gravino, A.E., 2006. Leishmania DNA load and
cytokine expression levels in asymptomatic naturally infected dogs.
Veterinary Parasitology 142, 271–280.