Canine Leishmania spp. infection in two distinct foci of visceral and cutaneous leishmaniasis in Tunisia.

Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) infantum and L. major, respectively, are endemic in Tunisia. The aim of the study was to assess canine Leishmania spp. infection prevalence as well as to identify the Leishmania species involved in two well-documented and geographically distinct VL and ZCL foci. One hundred seventy-six dogs were randomly recruited in the VL focus of Sbikha-Zaghouan (n = 100) and the ZCL focus of Echrarda-Nasrallah (n = 76). Physical examination and blood collection were systemically performed. Needle aspiration was done in case of lymph node (LN) enlargement. All sera were tested by ELISA. kDNA RT-PCR was performed on DNA extracts from (i) buffy coats of seropositive dogs and (ii) LN aspirates. Leishmania species identification was done by ITS1 PCR-sequencing. Thirty-three dogs (18.8%) were infected by Leishmania; 30 having anti-Leishmania antibodies and 3 were seronegative dogs with Leishmania DNA in LN aspirates. Prevalence of infection was significantly higher in VL foci than in ZCL foci (27% versus 7.9%, p = 0.002). Leishmania species was identified in 11 dogs and corresponded to L. infantum. Combination of serology and qPCR on LN aspirates seems to be the best option for canine leishmaniasis diagnosis. Infection is more frequent in VL foci and L. infantum is the only identified species.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Diagnosis of mediterranean visceral leishmaniasis by detection of leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device.

Current methods for diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures such as visceral aspiration and/or blood drawing. The use of diagnostic tests using oral fluid, which is easier to collect, would be more simple and practical for VL diagnosis, especially under field conditions. Oral fluids from 37 VL patients and 40 healthy controls were collected using Oracol devices. Blood samples and oral fluid specimens from both groups were analyzed by recombinant protein K39 (rK39) enzyme-linked immunosorbent assay and quantitative real-time PCR. Detection of antibodies in the oral fluid had a sensitivity of 100% and a specificity of 97.5%. Antibody levels measured in serum and oral fluid showed a significant positive correlation (ρ = 0.655 and P = 0.01). Detection of Leishmania DNA in oral fluid had a sensitivity of 94.6% and a specificity of 90%. The median parasite load estimated in blood was 133 parasites/ml (interquartile range [IR], 10 to 1,048), whereas that in oral fluid specimens was 3 parasites/ml (IR, 0.41 to 92). However, there was no significant linear relationship between parasite loads assessed in the two biological samples (ρ = 0.31 and P = 0.06). VL diagnosis based on specific antibody detection and Leishmania DNA identification using oral fluid samples was equivalent in accuracy to that using blood and therefore is promising for clinical use.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

Cutaneous Leishmaniasis in Algeria; Highlight on the Focus of M'Sila.

Algeria ranks second after Afghanistan for the incidence of cutaneous leishmaniasis (CL) worldwide. Here, we report a 34-years retrospective analysis of CL in Algeria and focused on the most affected region, the M'Sila province. All 66 cutaneous isolates corresponded to Leishmania (L.) major. Our study of the sandfly and rodent fauna further highlighted the high density of Phlebotomus papatasi and additional phlebotomine species of medical importance, not previously identified in M'Sila. Wild rodents belonging to nine species were trapped in M'Sila, and Psammomys obesus and Meriones shawi were found infected by L. major. In addition, Leishmania infantum was isolated from two visceral leishmaniasis cases, one dog and its proven vectors (P. perniciosus, P. longicuspis, and P. perfiliewi) inventoried during the survey. The high incidence of CL in the M'Sila province is likely a consequence of the increase in minimum temperatures recorded that constitutes suitable conditions for establishing a high endemicity and leads to an explosive rise in leishmaniases cases in this region. A thorough investigation of the underlying risk factors is urgently needed to detect new cases earlier. All these would improve the preparedness to fight the disease.

Development and Assessment of Leishmania major and Leishmania tropica Specific Loop-Mediated Isothermal Amplification Assays for the Diagnosis of Cutaneous Leishmaniasis in Tunisia.

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica, respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.

Unexpected diagnosis of basal cell carcinoma in a patient presenting with a secondary location of Leishmania parasites in the skin.

We report here a case of simultaneous cutaneous and visceral manifestations due to Leishmania L. infantum diagnosed in an immunocompetent adult. We describe a 74-year-old woman from Tunis, Tunisia, who presented a biologically confirmed visceral leishmaniasis infection concomitant with arm ulceration which appeared 2 years before. Leishmania DNA was detected by ITS PCR in both buffy coat and dermal scrapping of the arm lesion. Sequencing revealed that the 2 isolated strains corresponded to L. infantum and were 100% identical. The symptoms of visceral leishmaniasis responded to amphotericin B with rapid healing. However, the skin lesion did not improve although Leishmania PCR on dermal sample became negative. This location is probably secondarily to lymphatic or blood dissemination during the systemic visceral leishmaniasis infection. It would be favored by the inflammatory environment induced by the basal cell carcinoma subsequently diagnosed.

Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples.

Implementation of simple diagnostic tests using non-invasive collection of biological specimens is of great importance in the diagnosis of pediatric visceral leishmaniasis caused by Leishmania infantum. Latex agglutination kit (KAtex®) is widely used in the diagnosis mainly in L. donovani endemic areas. However its utilization in L. infantum endemic regions remains limited and its use on noninvasive biological specimen apart urine was not reported. In this study, KAtex® kit was used to detect Leishmania-related antigen in urine and oral fluid of 35 L. infantum visceral leishmaniasis cases and 62 controls including non-infectious disease and infectious disease controls (34 and 28 respectively). Sensitivity and specificity of urine based KAtex® were 51.4% and 98.3% respectively, whereas, sensitivity and specificity of oral-fluid based KAtex® were 80% and 88.3% respectively. Although, sensitivity of oral-fluid KAtex® was high, its specificity varied significantly according to the presence or the absence of an infectious disease (71.4% versus 97%, p=0.01).

Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas.

Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood. The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs' status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls. Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age <6months) and 5 old dogs (median age=72months, IQR: 45-84 months) that didn't respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn't respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2). The results of this study showed that IGRA is a useful new tool that can assess exposure to Leishmania in dogs with no to mild clinical signs in endemic area. Further comparative investigations using assays exploring cellular immunity are needed to determine its accuracy.