Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Meteorological, demographic, and environmental risk factors for Leishmania infantum in wildlife.

Leishmania infantum is an important and neglected vector-borne zoonotic protozoa endemic in the Mediterranean basin. Several wild and domestic mammals can contribute to maintaining its circulation but their importance as effective reservoirs is still under discussion and varies depending on local ecological communities. By combining environmental, climatic, and individual information, this study assessed the presence of L. infantum DNA in a set of wild species from Northwestern Italy and the potential ecological factors related to the risk of infection. From 2020 to 2022, 304 free-ranging wild animals were analyzed for the detection of L. infantum DNA in the spleen and popliteal lymph node (when available). The prevalence obtained in wild boar (Sus scrofa) and roe deer (Capreolus capreolus) was higher than those previously reported (% ± confidence interval 95%; 42.9 ± 18.4% and 27 ± 6.6% in wild boar and roe deer, respectively), and this is the first report of this parasite infecting the coypu Myocastor coypus (60 ± 34.7%). L. infantum DNA was detected in all the seasons including those free of adult sandflies and seasonal differences were minimal, suggesting a long course of infection. The models revealed that animals from rainy areas with higher greenness during the summer, highly populated by humans and predominantly covered by water surfaces had a higher risk of L. infantum. This study contributes to confirming previous findings on the existence of a sylvatic cycle for L. infantum in certain regions of Italy, as well as on the potential epidemiological role of roe deer for this parasite given the elevated prevalence found.

Feline leishmaniasis in an animal shelter in northeastern Brazil: Clinical aspects, coinfections, molecular detection, and serological study of a new recombinant protein.

Infection and clinical cases of leishmaniasis caused by Leishmania infantum in cats have been increasingly reported in several countries, including Brazil. In this study, we used an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) based on a recombinant antigen (rKDDR-plus) to detect anti-Leishmania antibodies in cats from an animal shelter in northeastern Brazil. We compared the results with an ELISA using L. infantum crude antigen (ELISA-CA). We also investigated the presence of Leishmania DNA in blood or ocular conjunctival samples as well as the association between Leishmania PCR positivity and serological positivity to feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Concerning serological assays, a higher positivity was detected using the ICT-rKDDR-plus (7.5%; 7/93) as compared to ELISA-rKDDR-plus (5.4%; 5/93) and ELISA-CA (4.3%; 4/93). Upon PCR testing, 52.7% (49/93) of the ocular conjunctival swabs and 48.3% (44/91) of the blood samples were positive. Together, PCR and serological testing revealed overall positivities of 73.1% (68/93) and 12.9% (12/93), respectively. Among PCR-positive samples, 45.5% (31/68) showed co-infection with FIV, 17.6% (12/68) with FeLV, and 82.3% (56/68) with T. gondii. More than half of the PCR-positive cats showed at least one clinical sign suggestive of leishmaniasis (58.8%; 40/68) and dermatological signs were the most frequent ones (45.5%; 31/68). Both tests employing the recombinant antigen rKDDR-plus (i.e., ICT-rKDDR-plus and ELISA-rKDDR-plus) detected more positive cats than the ELISA-CA but presented low overall accuracy. PCR testing using either blood or ocular conjunctival samples detected much more positive cats than serological tests.

A Geomedical Survey: Is There an Association Between Climatic Conditions and Leishmania Species Distribution in Iran During the Years 1999-2021?

PURPOSE: Iran is among the high-risk leishmaniasis regions in the world. WHO recommends the use of GIS as an ideal tool for healthcare authorities to predict the evolution of a disease, delimit the risk of outbreaks and identify critical areas. The aim of this research is to find the association between the main species of Leishmania (L. major, L. tropica, L. infantum) dispersion and climatic variables in Iran. METHODS: All molecular-based reports of leishmaniasis from Iran between 1999 and 2021 were gathered from reliable medical sources. Meteorological data (air and soil temperatures, annual rainfall and humidity) of the country along the study period were obtained from the Iranian Climatological Research Centre. The data concerning species distribution and climatic conditions during this period were moved to a base-map through raster layers using ArcGIS 10.4.1 software. The relationship between parasitological and climatic models was examined using ANOVA. RESULTS: High risk area maps, based on the cut-off thresholds, were generated for Leishmania major, L. tropica and L. infantum. According to the molecular-based reports, the L. major distribution was significantly related to all climatic variables, while L. tropica was merely related to rainfall and humidity, and the L. infantum distribution was significantly associated with rainfall, soil and air temperatures. CONCLUSION: The association between climatic conditions and Leishmania species distribution in Iran has been confirmed. Consequently, both, the relationship between climatic conditions and the geographical distribution of Leishmania species, and the use of GIS to better understand the spatial epidemiology of leishmaniasis, have been reaffirmed.

Leishmania infantum surveillence on dogs from Primavera do Leste, Mato Grosso state, Brazil.

The objective of this research was to determine the seroprevalence of Leishmania infantum in dogs in the city of Primavera do Leste, Mato Grosso, Brazil. A total of 109 serological samples from dogs were analyzed, collected between August 2021 to March 2022, using rapid immunochromatographic test, TR DPP® (LVC BioManguinhos), and Immunoenzymatic Assay (ELISA), EIE-Canine Visceral Leishmaniasis (Bio-Manguinhos®, Fiocruz). IgG anti-Leishmania infantum antibodies were detected 4.6% (5/109) by DPP test. Regardless of results, all samples were submitted to ELISA test, of those 0.9% (1/109) was positive, the same having also been positive for the DPP, from asymptomatic, adult dog living in the urban area. This is the first autochthonous report of CVL in the municipality. Since Visceral leishmaniasis caused by L. infantum is a disease of One Health concern, the results obtained are essential to fill the epidemiological gap of in the region. Furthermore, the health authorities should implement surveillance actions in order to monitoring of dogs, as well as survey of sand flies, and health education programs to stimulate the prevention of the disease.

Increased levels of cortisol are associated with the severity of experimental visceral leishmaniasis in a Leishmania (L.) infantum-hamster model.

BACKGROUND: Several infectious diseases are associated with hypothalamic-pituitary-adrenal (HPA) axis disorders by elevating circulating glucocorticoids (GCs), which are known to have an immunosuppressive potential. We conducted this study in golden hamsters, a suitable model for human visceral leishmaniasis (VL), to investigate the relationship of Leishmania (L.) infantum infection on cortisol production and VL severity. METHODS: L. infantum-infected (n = 42) and uninfected hamsters (n = 30) were followed-up at 30, 120, and 180 days post-infection (dpi). Plasma cortisol was analyzed by radioimmunoassay and cytokines, inducible nitric oxide synthase (iNOS), and arginase by RT-qPCR. RESULTS: All hamsters showed splenomegaly at 180 dpi. Increased parasite burden was associated with higher arginase expression and lower iNOS induction. Cortisol levels were elevated in infected animals in all-time points evaluated. Except for monocytes, all other leucocytes showed a strong negative correlation with cortisol, while transaminases were positively correlated. Immunological markers as interleukin (IL)-6, IL-1ß, IL-10, and transforming growth-factor-ß (TGF-ß) were positively correlated to cortisol production, while interferon-γ (IFN-γ) presented a negative correlation. A network analysis showed cortisol as an important knot linking clinical status and immunological parameters. CONCLUSIONS: These results suggest that L. infantum increases the systemic levels of cortisol, which showed to be associated with hematological, biochemical, and immunological parameters associated to VL severity.

Leishmania infantum xenodiagnosis from vertically infected dogs reveals significant skin tropism.

BACKGROUND: Dogs are the primary reservoir for human visceral leishmaniasis due to Leishmania infantum. Phlebotomine sand flies maintain zoonotic transmission of parasites between dogs and humans. A subset of dogs is infected transplacentally during gestation, but at what stage of the clinical spectrum vertically infected dogs contribute to the infected sand fly pool is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined infectiousness of dogs vertically infected with L. infantum from multiple clinical states to the vector Lutzomyia longipalpis using xenodiagnosis and found that vertically infected dogs were infectious to sand flies at differing rates. Dogs with mild to moderate disease showed significantly higher transmission to the vector than dogs with subclinical or severe disease. We documented a substantial parasite burden in the skin of vertically infected dogs by RT-qPCR, despite these dogs not having received intradermal parasites via sand flies. There was a highly significant correlation between skin parasite burden at the feeding site and sand fly parasite uptake. This suggests dogs with high skin parasite burden contribute the most to the infected sand fly pool. Although skin parasite load and parasitemia correlated with one another, the average parasite number detected in skin was significantly higher compared to blood in matched subjects. Thus, dermal resident parasites were infectious to sand flies from dogs without detectable parasitemia. CONCLUSIONS/SIGNIFICANCE: Together, our data implicate skin parasite burden and earlier clinical status as stronger indicators of outward transmission potential than blood parasite burden. Our studies of a population of dogs without vector transmission highlights the need to consider canine vertical transmission in surveillance and prevention strategies.

The sand fly (Diptera: Psychodidae) fauna of the urban area of Lassance, Northeast Minas Gerais, Brazil.

The present study aimed to check the sand flies' fauna on the municipality of Lassance, Minas Gerais, Brazil and detect the presence of Leishmania DNA on the female captured and determine the risk areas of the municipality. Sand flies were collected monthly from May 2018 to April 2019 using automatic light traps for 3 consecutive nights. Eight houses were selected as sample points due its previous reports of tegumentary leishmaniasis and/or canine leishmaniasis. The sand fly's fauna found on the present study it's represented by several medical importance species and the most abundant species found were Lutzomyia longipalpis (77.09%) and Nyssomyia intermedia (10.06%). Leishmania infantum DNA was detected in a pool of Lu. longipalpis resulting on a 2.81% of infection rate. By the frequency of the two most abundant species on this study, we developed a risk area map and it draws attention to sample point 6 due to disparate abundance of sand flies at this site (81.81%). Statistical overview shows Lu. longipalpis as dominant species and, still, Non-Metric Multidimensional Scaling analysis reveal high similarity on fauna's diversity on the study area. Our findings suggest that the diversity of sand flies from the municipality of Lassance may promote the circulation of Leishmania infantum parasites putting in risk the habitants and other mammal's species. Still, our study reinforces the necessity of specific studies focused on breed sites of phlebotomine and its' ecology to expand the knowledge about the behaviour of this group of insects applying directly to leishmaniases' epidemiology.

Passive case detection for canine visceral leishmaniasis control in urban Brazil: Determinants of population uptake.

BACKGROUND: In Brazil, the transmission of Leishmania infantum in urban settings is closely related to infection among dogs, with occasional transmission to humans. Serological screening of dogs for Leishmania spp. infection on requests of their owners (passive case detection) represents a frequent, but little studied, practice within the scope of Brazilian public health. This study identified factors associated with canine visceral leishmaniasis (CVL) diagnosis-seeking behavior of dog owners in Rondonópolis (236,000 inhabitants), a municipality in Central-Western Brazil where VL is endemic. Also, we evaluated the profile of dog owners and their animals screened on free demand. METHODOLOGY/PRINCIPAL FINDINGS: Using mixed effects negative binomial regression, we modelled the number of dogs screened for Leishmania infection on free demand per neighborhood from 2011 to 2016 as a function of time-dependent predictors (current or recent canine seropositivity and human VL incidence), distance to the screening site, and demographic variables. We assessed potential delays in the effect of time-dependent predictors on the outcome. Among 12,536 dogs screened for Leishmania infection, 64.2% were tested during serosurveys and 35.8% were tested on free demand. Of these, 63.9% were positive. Uptake of screening under free demand was strongly associated with higher levels of canine seropositivity in the neighborhood (current or recent) and decreasing distance to the screening site. A subsample of dog owners (n = 93) who sought CVL screening between 2016 and 2017 were interviewed in more detail. Owners with better socioeconomic status and dogs with apparent CVL clinical manifestations prevailed among them. CONCLUSIONS/SIGNIFICANCE: To support timely CVL management, passive case detection along with awareness activities aimed at dog owners should be encouraged in endemic areas. Screening sites should be prioritized in accessible zones, as well as in socio-economically disadvantage areas. In parallel, CVL active case detection should be continued as a surveillance tool to guide control actions.

Revisiting the heterogeneous global genomic population structure of Leishmania infantum.

Leishmania infantum is the main causative agent responsible for visceral leishmaniasis (VL), a disease with global distribution. The genomic structure and genetic variation of this species have been widely studied in different parts of the world. However, in some countries, this information is still yet unknown, as is the genomic behaviour of the main antigens used in VL diagnosis (rK39 and rK28), which have demonstrated variable sensitivity and specificity in a manner dependent on the geographic region analysed. The objective of this study was to explore the genomic architecture and diversity of four Colombian L. infantum isolates obtained in this study and to compare these results with the genetic analysis of 183 L. infantum isolates from across the world (obtained from public databases), as well as to analyse the whole rK39 and rK28 antigen sequences in our dataset. The results showed that, at the global level, L. infantum has high genetic homogeneity and extensive aneuploidy. Furthermore, we demonstrated that there are distinct populations of L. infantum circulating in various countries throughout the globe and that populations of distant countries have close genomic relationships. Additionally, this study demonstrated the high genetic variability of the rK28 antigen worldwide. In conclusion, our study allowed us to (i) expand our knowledge of the genomic structure of global L. infantum; (ii) describe the intra-specific genomic variability of this species; and (iii) understand the genomic characteristics of the main antigens used in the diagnosis of VL. Additionally, this is the first study to report whole-genome sequences of Colombian L. infantum isolates.

Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities.

BACKGROUND: Leishmania tarentolae is a protozoan isolated from geckoes (Tarentola annularis, Tarentola mauritanica), which is considered non-pathogenic and is transmitted by herpetophilic Sergentomyia spp. sand flies. This species occurs in sympatry with Leishmania infantum in areas where canine leishmaniasis is endemic. In the present study, we investigated the circulation of L. tarentolae and L. infantum in sand flies, dogs and lizards in a dog shelter in southern Italy, where canine leishmaniasis by L. infantum is endemic. METHODS: Sheltered dogs (n = 100) negative for Leishmania spp. (March 2020) were screened by immunofluorescence antibody test (IFAT) using promastigotes of both species at two time points (June 2020 and March 2021). Whole blood from dogs, tissues of Podarcis siculus lizards (n = 28) and sand flies (n = 2306) were also sampled and tested by a duplex real-time PCR (dqPCR). Host blood meal was assessed in sand flies by PCR. RESULTS: Overall, 16 dogs became positive for L. infantum and/or L. tarentolae by IFAT at one or both sampling periods. One canine blood sample was positive for L. infantum, whilst two for L. tarentolae by dqPCR. At the cytology of lizard blood, Leishmania spp. amastigote-like forms were detected in erythrocytes. Twenty-two tissue samples, mostly lung (21.4%), scored molecularly positive for L. tarentolae, corresponding to 10 lizards (i.e., 35.7%). Of the female Sergentomyia minuta sampled (n = 1252), 158 scored positive for L. tarentolae, four for L. infantum, and one co-infected. Two Phlebotomus perniciosus (out of 29 females) were positive for L. tarentolae. Engorged S. minuta (n = 10) fed on humans, and one P. perniciosus, positive for L. tarentolae, on lagomorphs. CONCLUSIONS: Dogs and lacertid lizards (Podarcis siculus) were herein found for the first time infected by L. tarentolae. The detection of both L. tarentolae and L. infantum in S. minuta and P. perniciosus suggests their sympatric circulation, with a potential overlap in vertebrate hosts. The interactions between L. tarentolae and L. infantum should be further investigated in both vectors and vertebrate hosts to understand the potential implications for the diagnosis and control of canine leishmaniasis in endemic areas.

Co-Circulation of Phleboviruses and Leishmania Parasites in Sand Flies from a Single Site in Italy Monitored between 2017 and 2020.

Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.

Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. METHODOLOGY/PRINCIPAL FINDINGS: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. CONCLUSIONS/SIGNIFICANCE: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.

Early onset of clinical leishmaniosis in a litter of pups with evidence of in utero transmission.

BACKGROUND: Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum. Although usually transmitted by phlebotomine sand flies, infection by vertical transmission and by blood transfusion have also been reported. METHODS: We describe the very early onset of clinical leishmaniosis, starting from 2 months of age, in a litter of pups born to an infected dam and sire. Seven pups from the litter of nine living in different households showed alopecic, exfoliative dermatitis and ulcerative cutaneous lesions. All pups and both parents were tested on at least one occasion both serologically, by enzyme-linked immunosorbent assay (ELISA), and by polymerase chain reaction (PCR) targeting the Leishmania ribosomal operon internal transcribed spacer 1 region and a short fragment of the kinetoplast minicircle; positive amplicons were sequenced. RESULTS: All nine pups were PCR positive for L. infantum verified by DNA sequencing, seven were positive by conjunctival, five by blood, four by lymph node, and one by skin PCR from an ulcerative lesion. Both pups with no clinical signs were seronegative, while five of the seven pups with dermatologic abnormalities were seropositive by ELISA. The sire had typical clinical dermatologic and visceral findings of CanL, was seropositive and PCR positive for L. infantum in the lymph node and fluid from the vas deferens tested after the testes were removed by castration. The dam was sub-clinically infected and seronegative, but positive by blood, lymph node and conjunctival PCR for L. infantum. Allopurinol administered to all clinically affected dogs resulted in clinical recovery. CONCLUSIONS: Infection with L. infantum in both parents, the very early age of clinical onset among most of the pups, and the fact that the puppies were born and detected with signs of leishmaniosis in the winter, which is a season without sand fly activity in Israel, strongly suggest vertical transmission. Awareness of the possibility of vertical transmission of L. infantum and infection in littermates should be increased. It is recommended that littermates of young dogs with clinical leishmaniosis should be tested for sub-clinical infection as they may also be infectious to sand flies and thus to other dogs and to humans. Restricting the mating of infected bitches should also be considered to prevent the vertical transmission of the infection.

Characterisation of the Leishmania donovani/L. infantum Hybrid Isolated from an Autochothonous Kala-Azar Patient: Preliminary Results of an In Vivo Model

Objective: In the present study, preliminary outcomes of the in vivo assessment of a Leishmania donovani/L. infantum hybrid isolated from a hospitalised patient with visceral leishmaniasis in Manisa and identified through analysis of the Leishmania-specific ITS-1, hsp70 and cpb gene regions are presented in comparison with reference strains of L. donovani and L. infantum. Methods: Three different study groups [(SG); n=16 mice each] and a control group (n=8 mice) were established with female Balb/C mice weighing 25-30 g. Reference L. donovani (MHOM/IN/1980/DD8), reference L. infantum (MHOM/TN/1980/IP1) and a L. donovani/L. infantum hybrid (MHOM/TR/2014/CBVL-LI/ LD), stored in liquid nitrogen, were thawed, cultured and incubated at 25 °C. A 15-µL dose of 1x108/mL promastigotes of three strains was applied to the tail veins of mice in the SG. After the mice were sacrificed, the liver and spleen tissues were removed and stored for immunological, immunohistochemical and pathological analyses. Results: The presence of infection in the liver and spleen tissues of mice was detected both by a specific enzyme-linked immunosorbent assay test and from the recovery of Leishmania promastigotes from liver and spleen tissues in NNN medium. However, Leishmania amastigotes were not observed in the touch biopsy smears of livers or spleens in either of the SGs. In addition, no evidence of tissue damage was identified in the SGs after immunohistochemical staining (with antibodies against IL-9, CD-117, MBP, CD163, CD4, CD8 and CD31). Conclusion: The obtained results show that hybrid Leishmania and reference L. donovani and L. infantum strains reached the liver and spleens of Balb/C mice in SGs but were of no pathological consequence. Yet, these three Leishmania isolates caused skin lesions when applied subcutaneously in Balb/C mice in another study. The findings presented in this study will be reassessed upon completion of the project, once the final results are obtained.

Frequency of detection and load of amastigotes in the pancreas of Leishmania infantum-seropositive dogs: clinical signs and histological changes.

BACKGROUND: Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and is highly lethal in humans and dogs if left untreated. The frequency of this parasite and associated histological changes in the pancreas of dogs are poorly studied. Therefore, the objectives of this study were to evaluate the frequency of detection and load of amastigotes in the pancreas of L. infantum-seropositive dogs and to identify the clinical signs and histological changes associated with parasitism of this organ. METHODS: One hundred forty-three dogs from an endemic area in Brazil that tested seropositive for L. infantum were studied. The dogs were clinically examined, killed, and necropsied between 2013 and 2014. One fragment of the pancreas was randomly collected for histopathology and immunohistochemistry, and spleen and bone marrow were collected for culture. RESULTS: Leishmania amastigotes were detected in the pancreas of 22 dogs (15.4%) by immunohistochemistry, all exhibiting L. infantum parasitism in the spleen and/or bone marrow. Poor body condition and cachexia were only associated with infection of the pancreas with Leishmania spp. (p = 0.021) and were found in 40.9% of dogs with pancreatic infection. Anorexia, vomiting, and/or diarrhea were observed in 9.2% of dogs with pancreatitis. The median parasite load in the pancreas was 1.4 infected macrophages/mm2. Pancreatic histological changes and their frequencies were: granulomatous pancreatitis (28.0%), lymphoplasmacytic pancreatitis (23.8%), acinar cell degeneration (6.3%), fibrosis (5.6%), hemorrhage (2.1%), eosinophilic pancreatitis (0.7%), suppurative pancreatitis (0.7%), and necrosis (0.7%). CONCLUSIONS: The present results demonstrate that L. infantum is one of the etiological agents of chronic pancreatitis in dogs; however, the frequency of detection and parasite load are low in this organ. The lack of an association of poor body condition and cachexia with pancreatitis and the low frequency of clinical signs commonly associated with pancreatitis suggest that a significant portion of the organ is not affected by this parasite. On the other hand, the association of poor body condition and cachexia with concomitant infection of the pancreas, spleen, and/or bone marrow with this parasite suggests that these manifestations are the result of a more advanced stage of canine visceral leishmaniasis.

Molecular and serological evaluation of zoonotic visceral leishmaniasis in dogs in a rural area of Fars province, southern Iran, as a source of Leishmania infantum infection.

Canine visceral leishmaniasis (CVL) is endemic in the southern parts of Iran. The current study aimed at molecular and serological evaluation of zoonotic visceral leishmaniasis in dogs in Fars province, southern Iran. Blood samples were collected from 60 dogs in the three villages in Fars Province. Serum samples were tested for antibodies against L. infantum by direct agglutination test (DAT). DNA was extracted from each dog's buffy coat and tested by PCR, targeting the Leishmania ITS-2 region. From a total of 60 studied dogs, 25 (41.7%) were female, and 35 (58.3%) were male. Dogs' age ranged from 1 to 7 years, with a mean age of 2.97 (±1.4) years. Anti-Leishmania antibodies were detected in sera samples of 28 (46.7%) dogs, (titre ≥ 1:320). Out of 28 seropositive cases, 13 (46.4%) were female, and 15 (53.6%) were male. Association between seropositivity and dogs' clinical signs was statistically significant (p < .05). Leishmania DNA was detected in the buffy coat of 3 of 60 studied dogs which were all seropositive by DAT. The PCR products were sequenced and molecular analysis showed that two of the isolates were Leishmania infantum, and one was L. tropica. The high proportion of seropositive dogs indicates the important role of these animals in the epidemiology of the disease in the region. Infected dogs with or without signs, especially those that are molecularly positive, can act as an active reservoir of the disease in the area.

Assessment of a recombinant protein from Leishmania infantum as a novel tool for Visceral Leishmaniasis (VL) diagnosis in VL/HIV co-infection cases.

Visceral Leishmaniasis and HIV-AIDS coinfection (VL/HIV) is considered a life-threatening pathology when undiagnosed and untreated, due to the immunosuppression caused by both diseases. Serological tests largely used for the VL diagnosis include the direct agglutination test (DAT), ELISA and immunochromatographic (ICT) assays. For VL diagnosis in HIV infections, different studies have shown that the use of the DAT assay facilitates the VL diagnosis in co-infected patients, since the performance of the most widely used ELISA and ICT tests, based on the recombinant protein rK39, are much less efficient in HIV co-infections. In this scenario, alternative recombinant antigens may help the development of new serological diagnostic methods which may improve the VL diagnosis for the co-infection cases. This work aimed to evaluate the use of the recombinant Lci2 antigen, related to, but antigenically more diverse than rK39, for VL diagnosis in co-infected sera through ELISA assays. A direct comparison between recombinant Lci2 and rK39 was thus carried out. The two proteins were first tested using indirect ELISA with sera from VL afflicted individuals and healthy controls, with similar performances. They were then tested with two different sets of VL/HIV co-infected cases and a significant drop in performance, for one of these groups, was observed for rK39 (32% sensitivity), but not for Lci2 (98% sensitivity). In fact, an almost perfect agreement (Kappa: 0.93) between the Lci2 ELISA and DAT was observed for the coinfected VL/HIV patients. Lci2 then has the potential to be used as a new tool for the VL diagnosis of VL/HIV co-infections.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.