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Leishmania (L.) infantum strains, isolated from varying hosts and clinical manifestations (cutaneous, visceral and canine leishmaniasis), were investigated in order to understand the genetic polymorphisms within this species in Algeria and Tunisia. Two DNA‐based typing methods were tested in order to evaluate their effectiveness against Multilocus enzyme electrophoresis (MLEE), widely considered as the reference method for Leishmania parasite typing. On the other hand, MLEE is cumbersome, high‐cost, time consuming and frequently does not detect intra‐species genetic polymorphisms. In this work, we used two molecular target regions to discriminate L. infantum strains, Internal transcribed spacer 1 (ITS1) and the cysteine proteinase B (cpb). The ITS1 region offers good resolution for Leishmania discrimination but does not spotlight intra‐species polymorphisms. In contrast, cpbE and cpbF PCR‐Sequencing demonstrated a certain variability within CL and VL Algerian and Tunisian L. infantum isolates. Following phylogenetic analyses of 44 L. infantum isolates, two main groups were identified, a group with 39 bp deletion in the cpb sequence, composed of cutaneous, visceral and canine isolates from both countries with no significant clinical or geographic distribution; these samples were typed as MON‐1, MON‐24, and MON‐80 zymodemes. A second group which presents a clear clusterization of Tunisian cutaneous strains belonging to the L. infantum MON‐24. This group, with no deletion in the mature domain of the cpb gene sequence, should be further explored with a higher number of samples. [ABSTRACT FROM AUTHOR]

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum. [ABSTRACT FROM AUTHOR]

Background: Discovered by Nicolle and Comte in 1908 in Tunisia, Leishmania infantum is an intracellular protozoan responsible for zoonotic canine leishmaniosis (CanL) and zoonotic human visceral leishmaniasis (HVL). It is endemic in several regions of the world, including Tunisia, with dogs considered as the main domestic reservoir. The geographic expansion of canine leishmaniosis (CanL) has been linked to global environmental changes that have affected the density and the distribution of its sand fly vectors. Methodology/Principal findings: In this study, a cross-sectional epidemiological survey on CanL was carried out in 8 localities in 8 bioclimatic areas of Tunisia. Blood samples were taken from 317 dogs after clinical examination. Collected sera were tested by indirect fluorescent antibody test (IFAT; 1:80) for the presence of anti-Leishmania infantum antibodies. The overall seroprevalence was 58.3% (185/317). Among positive dogs, only 16.7% showed clinical signs suggestive of leishmaniosis. Seroprevalence rates varied from 6.8% to 84.6% and from 28% to 66% by bioclimatic zone and age group, respectively. Serological positivity was not statistically associated with gender. The presence of Leishmania DNA in blood, using PCR, revealed 21.2% (64/302) prevalence in dogs, which varied by bioclimatic zone (7.3% to 31%) and age group (7% to 25%). The entomological survey carried out in the studied localities showed 16 species of the two genera (Phlebotomus and Sergentomyia). P. perniciosus, P. papatasi, and P. perfiliewi were the most dominant species with relative abundances of 34.7%, 25% and 20.4%, respectively. Conclusions/Significance: The present report suggests a significant increase of CanL in all bioclimatic areas in Tunisia and confirms the ongoing spread of the infection of dogs to the country's arid zone. Such an expansion of infection in dog population could be attributed to ecological, agronomic, social and climatic factors that affect the presence and density of the phlebotomine vectors. Author summary: Visceral leishmaniasis caused by Leishmania infantum is an endemic disease in several regions of the world, especially, in the Mediterranean basin, where dogs constitute the main domestic reservoir for humans. This zoonotic vector-borne disease is closely associated with the environment. We carried out studies in 8 different regions of Tunisia, collecting and testing blood samples from 317 dogs. Out of which, more than half (58.3%) tested positive for L. infantum (IFAT+), although, only one-third of these displayed clinical signs. However, the detection of Leishmania DNA by blood PCR revealed a global prevalence in dogs of 21.2% (64/302) with a variation between bioclimatic zones. Our entomological survey revealed 16 sand fly species, dominated by the Phlebotomus and Sergentomyia genera, of which the most dominant were P. perniciosus, P. perfiliewi and P. papatasi. The first two of these are recognized vectors of L. infantum. Canine leishmaniosis continues to spread across Tunisia, moving southward, to arid zones because of global warming and agronomic and social factors affecting the presence and density of the phlebotomine vectors. [ABSTRACT FROM AUTHOR]

Ten monoclonal microalgal cultures were obtained from several Tunisian inland water bodies, and their dichloromethane and methanolic extracts were screened for antibacterial, antileishmanial, and antioxidant properties, as well as phycobiliprotein production capacity. Cylindrospermopsis raciborskii has been shown to synthesize high levels of phycocyanin and may be an effective alternative source to other sources used for commercial production of phycocyanin. Chroococcus sp. and Leptolyngbya sp1. exhibited the strongest radical scavenging activity against DPPH (IC50 = 212.15 and 263.91 μg ml−1, respectively), indicating their promising potential for use as new effective and non-toxic antioxidants. Furthermore, Dunaliella sp. showed an interesting antileishmanial activity against the pathogens Leishmania infantum and Leishmania major (IC50 = 151 and 284 μg ml−1, respectively), thus representing a good candidate for use against cutaneous and visceral leishmaniasis in Tunisia, a country endemic to these diseases where thousands of new cases are registered every year. These results suggest that the strains of microalgae featured in this work have the potential to serve as natural alternative, safe and sustainable sources of high value-added products that could be used to improve the final biomass value. [ABSTRACT FROM AUTHOR]

Background: Zoonotic visceral leishmaniasis (VL) is endemic in the Mediterranean basin. However, large-scale comparative analyses of the commercial kits for the serological diagnosis of this neglected disease are lacking. This study compared the performances of four enzyme-linked immunosorbent assays (ELISA) and two immunochromatographic tests (ICT) as screening tests for the serodiagnosis of human VL in the Mediterranean region. Methodology/Principal findings: Serum samples from 319 patients living in France, Tunisia or Morocco were tested using two ICT (IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four ELISA reagents (NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania). The population with proven VL (n = 181) included 65 immunocompromised patients. Significantly higher percentages of false-negative results were obtained with all assays in immunocompromised patients, compared with the immunocompetent population. In the whole population, sensitivity and specificity ranged from 80.7% to 93.9% and from 95.7% to 100%, respectively. The maximum accuracy was observed with the Bordier and Vircell ELISA kits (96.2%), and the lowest accuracy with Ridascreen reagent (88.7%). New thresholds of positivity are proposed for the Bordier, Vircell and NovaLisa ELISA kits to achieve 95% sensitivity with the highest possible specificity. Western blot (WB), used as a confirmation method, showed 100% sensitivity and identified 10.1% of asymptomatic carriers among the control population from the South of France. Conclusions/Significance: This is the first study that compared commercially available kits for VL serodiagnosis in the endemic region of the Mediterranean basin. It provides specific information about the tests' performance to help clinicians and biologists to select the right assay for VL screening. Author summary: Human visceral leishmaniasis (VL) is a neglected disease endemic in the Mediterranean region, caused by Leishmania infantum. Accurate VL diagnosis is critical for rapid and correct patient management. Various serological tests are used, including commercial kits that require qualifications with specific information about their performance. In this study, we compared the performance of six commercial kits, two immunochromatographic tests (ICT; IT LEISH and TruQuick LEISH IgG/IgM Meridian) and four enzyme-linked immunosorbent assays (ELISA; NovaLisa Leishmania infantum IgG, Bordier Leishmania infantum, Ridascreen Leishmania IgG, and Vircell Leishmania), available in France and used as screening tests. The Bordier and Vircell ELISA reagents displayed the highest analytic performance compared with all the other kits. However, no test displayed 95% sensitivity at the thresholds recommended by the manufacturers. To reach this sensitivity value with the highest possible specificity, new thresholds are proposed for the Bordier, Vircell and NovaLisa ELISA reagents. Positive and negative results obtained with the six screening tests were confirmed by Western blot (100% sensitivity). Overall, this study presents a comparative analysis of commercially available kits for the serodiagnosis of human VL and provides additional information on the prevalence of asymptomatic carriers in the Mediterranean basin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phlebotomus perniciosus is one of the major vectors of Leishmania infantum in the Mediterranean basin. The aim of this work was (i) to provide information about abundance and temporal dynamics of this Larroussius species in a hot spot area of visceral leishmaniasis in Tunisia, (ii) to detect L. infantum DNA in wild caught female sandflies and (iii) to measure Phlebotomus perniciosus infection rate throughout the active season. Sandflies were collected monthly during one year using CDC miniature light-traps in house and in animal shelters. Male specimens were identified at species level according to morphological characters. Female specimens were conserved individually for molecular study. Leishmania infection was tested by kinetoplast DNA real-time PCR and ITS-1 PCR-sequencing. Subsequent sandfly species identification of infected specimens was done by mitochondrial cytochrome b sequencing. In one year period, overall 4,441 specimens (2230 males and 2211 females) were collected. Sandfly activity started in end-April and ended in early-November. Mean sandfly density in house was significantly lower than in animal shelters (51 ± 50 versus 504 ± 460 sandflies /CDC night, p<0.05). However, a higher proportion of females was found in house (58.4% versus 49.2%, p<0.001). Based on species identification of male specimens, Phlebotomus perniciosus was the dominant species (56% of the whole male sandfly fauna, p<0.0001). It showed two peaks of density in the active season, a sharp one in early May and a higher long lasting one from end-July to end-September. DNA was extracted from 190 female specimens randomly sampled and corresponding to 96 specimens from house and 94 from animal shelters. Twenty four female sandfly were infected by Leishmania infantum. All infected specimens were recognized as Phlebotomus perniciosus. Leishmania infantum infection rate in female sandflies was 2.3 fold higher in house than in animal shelters (17.7% versus 7.4%, p<0.05). In house, estimated number of infected specimens was the highest at the end of the active season. Abundance, dynamics of density and Leishmania infantum infection prevalence of Phlebotomus perniciosus in Tunisian hot spot of visceral leishmaniasis highlight the major role of this Phlebotominae species in L. infantum transmission. [ABSTRACT FROM AUTHOR]

Abstract: Over a period of ten years, a series of 694 Leishmania strains from Tunisian leishmaniasis foci were isolated and identified by isoenzymatic analysis. Strains were obtained from human cutaneous and visceral leishmaniasis in immunocompetent subjects, visceral leishmaniasis in imunocompromised individuals and from dogs with visceral leishmaniasis. Two classically dermotropic species, Leishmania (L.) major and Leishmania killicki were found. L. major with the single zymodeme MON-25 was the most isolated in cutaneous leishmaniasis foci of the Centre and South of Tunisia with a recent northern extension. L. killicki zymodeme MON-8 was sporadically found both in its classical microfocus of Tataouine in southeastern Tunisia as well as in some new foci in Southwestern, Central and Northern Tunisia. Leishmania infantum with its three zymodemes MON-1, MON-24 and MON-80 was isolated from both visceral and cutaneous human cases. The majority of L. infantum strains were found in the Northern part of the country; however, some strains were reported for the first time in the Southern part. L. infantum MON-1 was the only zymodeme isolated from canine leishmaniasis. [Copyright &y& Elsevier]

Background: Cutaneous leishmaniasis (CL) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France. Methods: A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180. Results: Ninety-two subjects were randomized. Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity. Conclusion: Application of WR279,396 for 20 days was found to be safe and effective in treating L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease. Trial Registration: ClinicalTrials.gov NCT00703924 Author Summary: Cutaneous leishmaniasis is due to a small parasite (Leishmania) that creates disfiguring sores, and affects more than one million persons (mainly children) each year. Treating lesions with a cream—instead of with injections as currently done—would greatly improve the well-being of affected patients. No cream formulation that would be efficient and would not create important skin irritation has been identified yet. Here, we tested a new cream formulation (WR279,396) containing paromomycin and gentamicin, two members of a well-known family of antibacterial antibiotics (aminoglycosides). Injectable paromomycin is efficient in other forms of the disease (visceral leishmaniasis). This was a carefully monitored study (phase 2) involving mainly children in Tunisia and France. The cream was applied twice a day for 20 days. The proportion of patients treated with the paromomycin-containing cream (active formulation) that cured (94%) was higher than that observed (71%) in patients treated with a cream that did not contain the active product (placebo formulation). Local irritation affected less than one-third of the patients and was usually mild. This new cream formulation was safe and effective in treating cutaneous leishmaniasis, thereby providing a new, simple, easily applicable, and inexpensive treatment for this neglected disease. [ABSTRACT FROM AUTHOR]

• Toscana virus and Leishmania infantum are co-circulating in Central Tunisia. • Phlebotomus perniciosus, Phlebotomus perfiliewi , and Phlebotomus longicuspis are the main vectors of Leishmania infantum and Toscana virus in irrigated areas located in arid Central Tunisia. • Environmental changes due to the development of irrigation systems in arid areas impact the emergence of zoonotic visceral leishmaniasis. • Human infection with Toscana virus and zoonotic visceral leishmaniasis represent serious public health problems in Tunisia. In the Mediterranean basin, sand flies are vectors of Leishmania parasites and phleboviruses affecting humans and animals. In this study, we aimed to investigate phlebovirus and Leishmania parasites circulating in a focus of zoonotic visceral leishmaniasis (ZVL) located in a highly irrigated area within the arid Central Tunisia, known mainly to be endemic for zoonotic cutaenous leishmaniasis (ZCL) caused Leishmania major and transmitted by Phlebotomus papatasi. Sand flies were collected using CDC light traps in the village of Saddaguia, an emergent focus of ZVL located in Central Tunisia during September-October 2014, 2015, and 2016. Pools of live female sand flies were screened for phleboviruses and Leishmania by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. Dead sand flies were identified morphologically to species level. Sand flies of the subgenus Larroussius mainly Phlebotomus perfiliewi, Phlebotomus perniciosus , and Phlebotomus longicuspis were predominant in this ZVL focus compared to P. papatasi. A total of 1932, 1740, and 444 sand flies were tested in 2014, 2015 and 2016, respectively. Pathogen screening performed on 4116 sand flies distributed in 148 pools revealed the presence of Leishmania infantum and Toscana virus. The minimum infection rates of sand flies with TOSV in 2014, 2015, and 2016 were 0.05%, 011%, and 0.22%, respectively. The minimum infection rates of sand flies with L. infantum in 2014, 2015, and 2016 were 0.25%, 012%, and 0.79%, respectively. No L. major was detected during the 3-years investigation in this ZVL focus. Our results showed clearly the endemic co-circulation of TOSV and L. infantum in this emergent ZVL focus. However, no co-infection of TOSV and L. infantum was detected in any of the sand fly pools investigated during the three years period. TOSV was isolated from positive pools in 2015. Phylogenetic analysis showed that the Tunisian strains of TOSV belonged to the sublineage A. Based on the present findings, our results provided strong evidence that TOSV and L. infantum are transmitted by the same predominant sand fly species of the subgenus Larroussius , and subsequently, humans and dogs could be co-infected through co-infected or successive infected bites. Our results showed clearly that the development of irrigation in arid areas contributed significantly to the establishment of stable transmission cycles of L. infantum and TOSV and subsequently to the emergence of a ZVL focus within this arid bio-geographical area characterized by the presence of multiple foci of ZCL located outside the study site. Thus, more studies are needed to better understand the impact of RNA viruses shared by vectors and reservoir hosts of L. infantum on the development of zoonotic visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: In Tunisia, asymptomatic carriage of Leishmania is poorly documented. Objective: The aim of the present study was to estimate the frequency of asymptomatic infection among the family members of patients with patent visceral leishmaniasis by using the Western blotting kit based on 14 and 16kDa bands. Material and methods: We tested 94 sera collected from 24 patients with patent visceral leishmaniasis and 70 from their families’ members. Results: The rate of seropositivity was 100% in the group of patients and 54.3% in the group of families’ members. The analysis of the Western blotting patterns showed that the 33kDa, 24kDa and to a lesser extent the 22kDa band were very indicative of patent visceral leishmaniasis in contrast to asymptomatic infection where these bands were very rarely detected. Conclusion: The results reported herein showed the high frequency of asymptomatic carriers of Leishmania among the families’ members of visceral leishmaniasis cases and the usefulness of the Western blotting as a screening technique and in distinguishing between patent visceral leishmaniasis and the asymptomatic carriage of Leishmania. [Copyright &y& Elsevier]