Leaf Decoction of Carica papaya Combined with Artesunate Prevents Recrudescence in Plasmodium berghei-Infected Mice.

Malaria treatment and control have become increasingly difficult because of the spread of drug-resistant strains of Plasmodium falciparum and Plasmodium vivax. Thus, there is a continuous need to develop new combination therapies such as artemisinin-based combination therapies (ACTs) to contrast the emergence of resistant Plasmodium strains. Despite ACT has been recommended by the World Health Organization since 2001, its overall deployment in poor endemic areas is very slow, principally due to its high cost. In the malaria endemic areas, plant remedies are still widely used mostly without assurance of their efficacy and/or safety. A variety of widespread herbal drugs or natural products were already reported for their possible plasmodicidal activities, but the studies concerning their activity in combination with artemisinins are very scarce. The antimalarial activity of papaya is mostly anecdotal, and the present study is aimed at investigating the antiplasmodial activity of a decoction obtained by traditional recipe from the mature leaves of Carica papaya. The decoction was analyzed by HPLC-DAD-MS (high performance liquid chromatography coupled with diodoarray detector and mass spectrometry) showing the presence of caffeoyl derivatives and di- and triglycosides of flavonols. The extract was found to be active against P. falciparum 3D7 strains with a synergism in the presence of artemisinin. In vivo activity against the murine malaria model of Plasmodium berghei was disclosed both for the dried extract alone (250, 500, and 750 mg/kg/d) and for its combination with artesunate (250 mg/kg/d papaya plus 10 mg/kg/d artesunate). This combination displayed the greatest antimalarial activity in terms of reduction of parasitemia and prevention of recrudescence in animals recovered from the infection.

Validation of a recombinant based antibody ELISA for diagnosis of human and canine leishmaniasis.

In this study, a recombinant chimeric antigen (CA) ELISA was validated as a single test for both human and dog leishmaniasis. Serum panels included 327 human and 339 canine IFAT-positive and 1113 human and 1078 canine IFAT-negative samples. CA-ELISA was carried out using the same serum dilution, and labelled protein A as secondary reagent. Test performances were calculated using ROC analysis. For the human panel, the test showed diagnostic accuracy (DA) 0.974, specificity (Sp) 97.12%, sensitivity (Se) 91.44%, and concordance (K) 0.88. The dog panel showed DA 0.998, Sp 99.54%, Se 98.54%, and K 0.98. The proposed method is the best recombinant antigen-based ELISA, and can be used as IFAT substitute for mass screening.

Nostril mass caused by Leishmania spp. in a dog.

Canine leishmaniasis (CanL) is a systemic zoonotic disease caused by the protozoan Leishmania, an intracellular macrophage parasite, transmitted by the bite of phlebotomine sandflies. In dogs, the clinical disease is mostly characterised by symptoms associated with viscerocutaneous lesions such as lymphadenopathy, splenomegaly, skin lesions, and renal and ocular disease caused by the deposition of immune complexes. The parasite may provoke mucosal lesions which cause atypical clinical signs. The aim of this study is to describe an atypical nostril mass in a dog infected by Leishmania. Clinical examination did not show any systemic clinical signs, while haematological, biochemical, and urinary parameters demonstrated a mild disease stage. Diagnosis was confirmed through the isolation of cultured live parasites by biopsy. The dog was treated with a combination of miltefosine and allopurinol, showing full remission of clinical symptoms after 2 months. The authors outline the importance of considering CanL in the differential diagnosis of mucous and tumour-like lesions.

Serological and molecular tools to diagnose visceral leishmaniasis: 2-years' experience of a single center in Northern Italy.

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL.

Epidemiological survey on Leishmania infection in red foxes (Vulpes vulpes) and hunting dogs sharing the same rural area in Southern Italy.

Southern Italy, particularly Campania region, is an area where canine leishmaniasis (CanL) and zoonotic human visceral leishmaniasis (VL) are endemic. The red fox (Vulpes vulpes) has been hypothesized to play a role in occurrence of CanL in Italy but specific studies are poor. The aim of the present survey was to investigate the prevalence of Leishmania infection in dogs and foxes living in the same rural area (Picentini hills). 123 sera from autochthonous fox-hunting dogs were examined by immunofluorescent-antibody test (IFAT) using a cut-off of 1:160. The seroprevalence of dogs examined was 17.9%. Moreover, 48 foxes were examined after having been shooted by hunters or road accidents. Spleen, liver and lymph node samples were analyzed by specific Leishmania nested PCR (n-PCR). 10 foxes were found infected by L. infantum (20.8%) of which 4 animals in spleen, 2 in lymph nodes and 4 both in spleen and lymph nodes. The overall n-PCR positivity was 17.4% for spleen samples and 13.3% for lymph nodes; all liver samples resulted negative. In positive PCR foxes no signs clearly referable to leishmaniasis were recorded at necropsy. The results confirmed the presence of L. infantum infection in red foxes from Southern Italy, with a moderate level of exposure. Because large proportions of dogs with ascertained progressive leishmaniasis show a prolonged "subpatent condition" during which they are only positive to n-PCR before seroconversion, our results allow to assume that exposure risk in foxes is lower than hunting dogs living in the studied area.

Epidemiology of Imported Leishmaniasis in Italy: Implications for a European Endemic Country.

In the past decade, the number of imported leishmaniasis cases has increased in countries of Western Europe. The trend is associated with increasing travels, ecotourism activity, military operations and immigration. While in endemic countries leishmaniasis is usually well diagnosed, accurate patient history and parasite identification are necessary to distinguish between autochthonous and imported cases. This is particularly important, as new Leishmania species/genotypes may be introduced and transmitted by local phlebotomine vectors without appropriate surveillance, with unpredictable consequences. We report on the surveillance of imported leishmaniasis performed by the Leishmania Identification Reference Centre of Rome from 1986 through 2012, involving health care centres from 16/20 Italian regions. Suspected imported cases were analyzed and conclusions were based on clinical, epidemiological and diagnostic findings. Over the years, different parasite identification methods were employed, including MultiLocus Enzyme Electrophoresis and molecular techniques combining disease diagnosis (SSU rDNA nested-PCR) and Leishmania typing (nuclear repetitive sequence and ITS-1 PCR-RFLPs). A total of 105 imported cases were recorded (annual range: 0-20) of which 36 were visceral (VL) (16 HIV-coinfections) and 69 cutaneous (CL) cases; 85 cases (52 CL) were from the Old World and 20 (17 CL) from the New World. Eight Leishmania species were identified, of which 7 were exotic to Italy. VL importation until 1995 was associated with the spread of Mediterranean Leishmania-HIV co-infections in early 1990s. Following the introduction of HAART treatment, such cases became occasional in Italians but relatively frequent among immigrants. In contrast, a steady increase of CL cases was observed from different areas of the Old and New Worlds, that in recent years included mainly immigrants 'visiting friends and relatives' and Italian tourists. This positive trend likely depends on better diagnosis and reporting; however, we suspect that many CL cases remained unrecognized. Given the relatively low incidence of leishmaniasis importation, the risk of introduction of exotic parasites appears limited, although the detection of anthroponotic species requires attention.

Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs. METHODOLOGY/PRINCIPAL FINDINGS: Sera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus. CONCLUSIONS: These results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

A randomised, double-blind, controlled efficacy trial of the LiESP/QA-21 vaccine in naïve dogs exposed to two leishmania infantum transmission seasons.

Canine leishmaniasis is an important zoonosis caused by uncontrolled infection with Leishmania infantum, where an inappropriate immune response is not only responsible for permitting this intracellular parasite to multiply, but is also responsible for several of the pathological processes seen in this disease. Effective canine vaccines are therefore a highly desirable prevention tool. In this randomised, double-blinded, controlled trial, the efficacy of the LiESP/QA-21 vaccine (CaniLeish, Virbac, France) was assessed by exposing 90 naïve dogs to natural L. infantum infection during 2 consecutive transmission seasons, in two highly endemic areas of the Mediterranean basin. Regular PCR, culture, serological and clinical examinations were performed, and the infection/disease status of the dogs was classified at each examination. The vaccine was well-tolerated, and provided a significant reduction in the risk of progressing to uncontrolled active infection (p = 0.025) or symptomatic disease (p = 0.046), with an efficacy of 68.4% and a protection rate of 92.7%. The probability of becoming PCR positive was similar between groups, but the probability of returning to a PCR negative condition was higher in the vaccinated group (p = 0.04). In conclusion, we confirmed the interest of using this vaccine as part of a comprehensive control program for canine leishmaniasis, and validated the use of a protocol based on regular in-depth assessments over time to assess the efficacy of a canine leishmaniasis vaccine.

Prospective study on the incidence and progression of clinical signs in naïve dogs naturally infected by Leishmania infantum.

The incidence of clinical and clinicopathological signs associated with the progression of infection was evaluated prospectively in 329 naïve young dogs exposed to Leishmania infantum transmission and examined periodically during 22 months (M). The dogs were part of Leishmania vaccine investigations performed under natural conditions. Vaccinated groups were considered in the evaluation when the vaccine resulted non-protective and the appearance and progression of signs did not differ statistically from controls at each time point, otherwise only control groups were included. 115 beagles were part of 3 studies (A to C) performed in the same kennel; 214 owned dogs (29 breeds, 2.3% beagles) were included in a study (D) performed in 45 endemic sites. At M22 the prevalence of any Leishmania infection stage classified as subpatent, active asymptomatic, or symptomatic was 59.8% in studies A-C and 29.2% in study D. Despite different breed composition and infection incidence, the relative proportion of active infections and the progression and type of clinical and clinicopathological signs have been similar in both study sets. All asymptomatic active infections recorded have invariably progressed to full-blown disease, resulting in 56 sick dogs at M22. In these dogs, lymph nodes enlargement and weight loss--recorded from M12--were the most common signs. Cutaneous signs were seen late (M18) and less frequently. Ocular signs appeared even later, being sporadically recorded at M22. Most clinicopathological alterations became evident from M12, although a few cases of thrombocytopenia or mild non-regenerative anemia were already observed at M6. Albumin/globulin inversions were recorded from M12 and urea/creatinine increase appeared mostly from M18. Altogether our findings indicate that any susceptible young dogs naturally infected by L. infantum present a common pattern of progression of signs during 2 years post infection, providing clues for medical and epidemiological applied aspects.

First record of autochthonous canine leishmaniasis in Hungary.

Hungary is traditionally regarded as a leishmaniasis-free country, and human or canine cases diagnosed locally have been recorded as imported. However, recent entomological surveys have verified the presence in Hungary of Phlebotomus neglectus and Phlebotomus perfiliewi perfiliewi, which have been incriminated as competent vectors of Leishmania infantum elsewhere in Europe. Following the occurrence in October 2007 of an undisputable clinical case of L. infantum canine leishmaniasis (CanL) in a 4-year-old female pug in a kennel of 20 dogs in Tolna province, an investigation was performed to assess the infection status in that canine population and to search for putative phlebotomine vectors. Another female pug became sick during the study period (May-November 2008) and L. infantum was confirmed as the causative agent. The other animals appeared clinically healthy; however, 4 additional dogs were found positive by indirect fluorescent antibody test (2 dogs), or by buffy-coat PCR (1 dog), or by both methods (1 dog). Hence the overall Leishmania infection prevalence in the kennel was 30% (6/20). All dogs were born in the same place and had been always kept outdoors. They had neither been abroad nor received a blood transfusion. No sand flies were collected with CDC Standard Miniature Light traps, Mosquito Magnet(®) X (MMX) dry ice-baited traps, or sticky traps placed either in or around the kennel and at nearby chicken yards during July and August of 2008 and 2009. Considering the dogs' historical background and the failure to trap any sand fly vectors in the kennel area, the origin of CanL in this site remains unexplained.

Longitudinal study on the detection of canine Leishmania infections by conjunctival swab analysis and correlation with entomological parameters.

A longitudinal study was carried out on kennelled stray dogs in a canine leishmaniasis (CanL) endemic area, to evaluate early and late diagnostic performance of a non-invasive conjunctival swab (CS) nested (n)-PCR analysis for Leishmania detection in 2 cohorts of dogs, respectively. (A) Sixty-five IFAT- and CS n-PCR-negative dogs exposed to, and followed up once or twice a month during a full sand fly season (July-November 2008). In parallel, a sand fly survey was performed on site using standard sticky traps set twice a month, for a cumulative surface of 63 m(2). (B) Seventeen IFAT- and CS n-PCR-negative dogs found positive in July 2008 at the peripheral blood buffy-coat (BC) n-PCR. These dogs were examined again by BC n-PCR in September and November 2008, and before the subsequent transmission season (May 2009) along with CS n-PCR and IFAT. None of the cohort (A) dogs converted to positive CS n-PCR during the transmission season. Although approximately 2500 phlebotomine specimens were collected with peaks of 100-147 specimens/m(2) sticky trap, the cumulative density of the only proven CanL vector in the area (Phlebotomus perniciosus) was found to be very low (0.5/m(2)). All cohort (B) dogs remained substantially seronegative; BC n-PCR showed an intermittent positive trend during the period surveyed, resulting in 82% conversions to negative by the end of the study, in contrast with 71% conversions to positive at the CS n-PCR analysis. In conclusion, while CS n-PCR was not found effective for the early detection of Leishmania contacts in dogs exposed to a low pressure of vectorial transmission, this assay showed to slowly convert to positive in a high rate of dogs, in the absence of seroconversion. CS n-PCR technique can be a suitable marker for assessing Leishmania exposure in dogs as a non-invasive alternative to current serological and molecular tools.

Risk assessment for canine leishmaniasis spreading in the north of Italy.

The incidence of zoonotic visceral leishmaniasis has not only been recognized but is, in fact, increasing in territories of northern continental Italy previously regarded as non-endemic. Recent findings of sporadic autochthonous canine infections and the presence of phlebotomine vectors in some provinces of north-eastern Italy have stimulated risk assessment for the spreading of leishmaniasis in the autonomous province of Bolzano-South Tyrol, the northernmost territory of the Italian eastern Alps. In July 2008, 61 phlebotomine sand flies (Diptera, Psychodidae) were caught and identified as Phlebotomus perniciosus and Sergentomyia minuta. This is the first record in South Tyrol of P. perniciosus, the most competent vector of Leishmania infantum in Mediterranean countries. Leishmania serology on local dogs kept in kennels gave negative results, while only imported canine leishmaniasis cases were reported by local veterinaries through a questionnaire survey. Bio-geographic aspects and epidemiological consequences are analyzed in relation with the risk of leishmaniasis introduction into the area.

Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-PCR techniques in a cohort of naive dogs exposed to three consecutive transmission seasons.

Most experience in the comparison of diagnostic tools for canine leishmaniasis comes from cross-sectional surveys of dogs of different ages and breeds and in cases with unknown onset and duration of leishmaniasis. A longitudinal study was performed on 43 beagle dogs exposed to three transmission seasons (2002 to 2004) of Mediterranean leishmaniasis and examined periodically over 32 months through bone marrow microscopy and nested PCR (n-PCR), lymph node culture, serology (immunofluorescent-antibody test), and evaluation of clinical parameters. Starting from January 2003, the highest rate of positives was detected by n-PCR at all assessments (from 23.3% to 97.3%). Sensitivities of serologic and parasitological techniques were lower but increased with time, from 15.8% to 75.0 to 77.8%. Some dogs that tested positive by n-PCR but negative by other tests ("subpatent infection") remained so until the end of the study or converted to negative in subsequent assessments, whereas all dogs with positive serology and/or microscopy/culture ("asymptomatic patent infection") exhibited progressive leishmaniasis; 68% of them developed clinical disease ("symptomatic patent infection") during the study, at 7 (range, 3 to 14) months after being positive to all tests. Postexposure infection incidences were high and were significantly different between 2002 and 2003 exposures (39.5% and 91.7%, respectively). The time course of infection was highly variable in each dog, with three patterns being identified: (i) rapid establishment of a patent condition (0 to 2 months from detection of infection); (ii) a prolonged subpatent condition (4 to 22 months) before progression; and (iii) a transient subpatent condition followed by 10 to 21 months of apparent Leishmania-negative status before progression.

Visceral leishmaniasis treatment, Italy.

First-line drug treatment was recorded in 573 immunocompetent patients with visceral leishmaniasis in Italy. In the past 12 years, the proportion of antimonial treatments decreased from 100% to 2.8%, while the proportion of amphotericin B treatments increased from 0% to 97.2%. The countrywide change in therapy is a response to both disease reemergence and increasing antimonial failure.

Mediterranean visceral leishmaniasis in HIV-negative adults: a retrospective analysis of 64 consecutive cases (1995-2001).

AIM: To evaluate in a retrospective analysis cases of Mediterranean visceral leishmaniasis (VL) diagnosed in HIV-negative adults during a 7-year period. MATERIALS AND METHODS: Demographic data, previous or underlying diseases, clinical and laboratory features and therapeutic findings were considered. RESULTS: A total of 64 patients were included, of whom 10 (16%) had underlying diseases and two were pregnant. Fever and hepatosplenomegaly were the main presenting symptoms, whereas pancytopenia and an elevated erythrocyte sedimentation rate were observed in all cases. Smears from bone marrow aspirate were positive at microscopy in 62 cases (97%). Twenty-four patients received meglumine antimoniate (MA) given during 21 consecutive days (20 mg/kg per day), and 40 patients liposomal amphotericin B (l-AmB) given at days 1-5 and 10 (3 mg/kg per day). Both groups' clinical and laboratory findings improved, but patients on l-AmB therapy had a faster recovery (85% on l-AmB therapy and 50% on MA therapy showed defervescence at day 5, P < 0.01). Treatment failures were observed in five cases, three (12%) on MA and two (5%) on l-AmB therapy. No significant toxicity was observed in patients treated with l-AmB, whereas three (12%) patients treated with MA showed electrocardiographic abnormalities. CONCLUSIONS: l-AmB therapy may be considered the treatment of choice for any adult patients with Mediterranean VL, since it permits a faster recovery, has a lower incidence of side effects and is useful also in immunosuppressed patients.