Clinical polymorphism of zoonotic cutaneous leishmaniasis: combination of the clinical and the parasitological diagnosis.

Zoonotic cutaneous leishmaniasis (ZCL) is a neglected tropical disease caused by Leishmania (L.) major. This zoonosis is characterized by a broad-spectrum clinical polymorphism and may be underestimated and poorly treated since it is a simulator of various dermatoses. The aim of our study was to analyze the clinical polymorphism of patients with ZCL. A total of 142 patients with confirmed CL based on the microscopic examination of skin lesion biopsies were included in this study. Molecular typing of Leishmania species revealed that all patients were infected with L. major. In total, 14 clinical forms were observed. Six were typical and eight were atypical. The typical ZCL forms are grouped as follows: papular (26.76%), ulcero-crusted (26.05%), ulcerated (13.38%), impetiginous (9.86%), nodular (9.15%), and papulo-nodular (5.63%) lesions. In atypical ZCL forms, we described erythematous (2.81%), erysipeloid (1.4%), sporotrichoid, (1.4%), keratotic (0.7%) lupoid (0.7%), lichenoid (0.7%), psoriasiform (0.7%), and zosteriform (0.7%) lesions. Here, the lichenoid and the keratotic forms caused by L. major were reported for the first time in Tunisia. These findings will help physicians to be aware of the unusual lesions of ZCL that could be confused with other dermatological diseases. For this reason, it will be necessary to improve the diagnosis of CL especially in endemic areas. Such large clinical polymorphism caused by L. major may be the result of a complex association between the vector microbiota, the parasite, and the host immune state, and further studies should be carried out in order to reveal the mechanisms involved in clinical polymorphism of ZCL.

On abnormal Phlebotomus perniciosus (Diptera: Psychodidae: Phlebotominae) from the center of Tunisia.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Identification of cuticle and midgut fungal microflora of phlebotomine sandflies collected in Tunisia.

Phlebotomine sand flies (Diptera: Psychodidae) are the proven vectors of Leishmaniases which are widespread parasitosis in many tropical and subtropical countries. The development of infective metacyclic Leishmania (Kinetoplastida: Trypanosomatidae) promastigotes stage is restricted to the vector midgut. Recently, several studies have assessed the influence of the sand fly midgut fungal microflora on the development of invective Leishmania stage. The aim of this study was to identify the fungal microflora from the cuticle and midgut of wild caught sandflies. A total of 50 sandflies were caught in two different leishmaniasis foci of center Tunisia and analyzed using an in vitro isolation of fungi followed by a morphological and molecular identification of fungal isolates. The morphological identification of sandflies specimens revealed five Species: Phlebotomus (P.) papatasi (n = 25), P. perniciosus (n = 15) P. riouxi (n = 6), P. longicuspis (n = 3) and P. sergenti (n = 1). Forty positive fungal cultures were isolated from 34 sand flies (19 males and 15 females) distributed as following: P. papatasi (n = 16), P. perniciosus (n = 11), P. riouxi (n = 4), P. longicuspis (n = 2) and P. sergenti (n = 1). Thirty-five cultures were isolated from the cuticles and five from the guts. A total of 15 fungi genera belonging to 8 families were identified with the predominance of Aspergillus genus followed by Penicillium genus. Among the 15 fungi genera, five were common between males and females specimens. Lecytophora canina and Leishmania major co-infection was detected in the gut of a female P. papatasi. Our preliminary findings highlight the high diversity of fungal microflora from the sand flies midguts.

Epidemiology of Pityriasis versicolor in Tunisia: Clinical features and characterization of Malassezia species.

Malassezia (M.) genus includes commensal yeasts of increasing medical importance, as they result in many diseases, ranging from pityriasis versicolor (PV) to systemic infections. Previous studies reported geographical variations in distribution of Malassezia species in PV lesions. The aims of the current study were to define the clinico-demographic features of PV in Tunisia, to characterize Malassezia isolates using phenotypic and molecular techniques and to find out any association between species and clinico-demographic parameters. In total, 120 PV patients were enrolled in this study. Skin scrapings were collected and inoculated on Sabouraud agar and modified Dixon medium. Malassezia species were identified using conventional phenotypic methods and 26 s rDNA PCR-RFLP. The highest prevalence of PV was observed among young adults' group. The most affected body areas were the back and neck. In overall, 50.8% and 35% of PV cases had pruritus and history of recurrence respectively. The overall concordance between phenotypic and molecular methods was high (80.95%). The discordant results are rather due to the presence of multiple species in a single culture than true misidentification. Using PCR-RFLP, M. furfur was the most isolated species (38.7%) followed by M. globosa (37.7%), M. restricta and M. sympodialis. No statistically significant association was noted between Malassezia spp. and clinico-demographic characteristics. Unlike many reports from temperate climate countries, M. furfur and M. globosa along together were the most frequently isolated species in Tunisian PV patients. Although phenotypic methods remain simple and cost-effective, molecular techniques are considered as fast and accurate methods for diagnosis purposes.

Morphological and molecular differentiation between Culicoides oxystoma and Culicoides kingi (Diptera: Ceratopogonidae) in Tunisia.

BACKGROUND: Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. METHODS: Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR-RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. RESULTS: Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR-RFLP method. CONCLUSIONS: This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR-RFLP method. This approach could be applied in future epidemiological studies at the national and international levels.

Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species.

BACKGROUND: Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs. METHODOLOGY AND FINDINGS: A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively. CONCLUSION: Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.

The vector competence of Phlebotomus perniciosus for Leishmania infantum zymodemes of Tunisia.

Experimental infections of Phlebotomus (L.) perniciosus from a colony established in Madrid (Spain) carried out with the Leishmania (L.) infantum zymodemes MON-1, MON-24, and MON-80 isolated in Tunisia are reported here. Laboratory-reared female sand flies were experimentally fed via membrane feeding device on a suspension of L. infantum promastigotes in defibrinated rabbit blood (107/ml). Engorged females were dissected at progressive time points postfeeding to observe the intravectorial cycle of different L. infantum zymodemes. Development in the sand fly midgut of L. infantum parasites to the infective metacyclic promastigotes and monitoring the forward progression of parasites to finally reach the stomodeal valve (SV) of the sand fly were assessed. All tested L. infantum zymodemes developed properly in P. perniciosus. Experimental feeding with suspensions of promastigotes of all zymodemes led to very heavy late-stage infections. MON-24 and MON-80 zymodemes colonized the (SV) of P. perniciosus earlier than zymodeme MON-1, 2 and 4 days, respectively. Metacyclic promastigotes were observed in all experimental infections. The study shows for the first time that colonized P. perniciosus is able to acquire, retain, and develop in its midgut the zymodemes MON-24 and MON-80 isolated in Tunisia and highlights the putative role of this sand fly species in the transmission of such zymodemes to mammalian hosts in this country. The ability of experimentally infected sand fly species to transmit by bite such zymodemes needs to be assessed.

Cutaneous leishmaniasis in northwestern Saudi Arabia: identification of sand fly fauna and parasites.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne disease transmitted by the bite of an infected sand fly. This disease is highly prevalent in Saudi Arabia where Leishmania major and L. tropica are the etiological agents. In the region of Hail, northwestern of Saudi Arabia, the incidence is about 183 cases/year. However, the epidemiology of the disease in this area is not well understood. Thus, an epidemiological survey was conducted in 2015-2016 to identify the circulating parasite and the sand fly fauna in the region of Hail. Skin lesion scrapings were collected from suspected patients with CL. METHODS: The diagnosis was made by microscopic examination of Giemsa-stained smear and PCR. The parasite was identified by PCR and sequencing of the single copy putative translation initiation factor alpha subunit gene. Sand fly specimens were collected and identified morphologically. Total DNA was extracted from the abdomen of female specimens and Leishmania DNA was detected by PCR. RESULTS: Among the 57 examined patients, 37 were positive for CL. The identification of the parasite has revealed the single species Leishmania major. The 384 sand flies were collected belonged to two genera (Phlebotomus and Sergentomyia), six sub-genera and six species. Phlebotomus papatasi, Ph. kazeruni and Sergentomyia clydei were the dominant species. Leishmania DNA was detected in two females of Ph. papatasi two of Ph. kazeruni and one specimen of Sergentomyia clydei. CONCLUSIONS: Leishmania major is confirmed to be the etiological agent of cutaneous leishmaniasis in northwestern Saudi Arabia. The molecular detection of Leishmania DNA in Ph. papatasi and Ph. kazeruni supports the potential role of these two species in the transmission of Leishmania. Further epidemiological studies are needed to prove their role and to evaluate the burden of CL in the study region.

Clinical Presentation of Cutaneous Leishmaniasis caused by Leishmania major.

BACKGROUND/AIMS: The diagnosis of cutaneous leishmaniasis (CL) is based on the microscopic detection of amastigote, isolation of the parasite, or the detection of Leishmania DNA. Nevertheless, since these techniques are time consuming and not usually available in many endemic countries, the diagnosis remains clinical. Consequently, such disease may be overlooked because of its similarity to other skin diseases. The aim of this study is to describe the clinical polymorphism of CL caused by Leishmaniamajor. METHODS: A cross-sectional survey was carried out on 166 patients. Diagnoses were made by both microscopic examination of stained tissue-scraping smears and PCR. The Leishmania species was identified by restriction enzyme analysis of the ribosomal internal transcribed spacer 1 region. The clinical polymorphism was analyzed only for patients with a positive diagnosis for CL and L. major as the identified species. RESULTS AND CONCLUSION: Of the 166 patients, 75 patients fit the inclusion criteria. Twelve different types of CL caused by L. major were defined. The most common type was the ulcero-crusted form followed by the papulonodular form and the impetigenous form. The ulcerated, mucocutaneous, lupoid, and sporotricoid forms were less common. The eczematiform, erysipeloid, verrucous, psoriasiform, and pseudotumoral types were represented by a single case. Zoonotic CL caused by L. major can simulate many other skin diseases, which may lead to a significant spread of this disease and increases in morbidity and drug resistance. This large polymorphism may be the result of a complex association between the genetics of the parasite and the immune response of the host.

Unexpected co-detection of promastigote and amastigote Leishmania forms in a human cutaneous lesion: implications for leishmaniasis physiopathology and treatment.

Cutaneous leishmaniasis pathogenicity depends on the survival and replication of the parasitic protozoa in the form of non-motile amastigotes inside macrophages. Here, we report the unprecedented observation of both Leishmania major amastigote and promastigote forms (the latter is normally detected only in the mid gut of the insect vector or in vitro culture) in a cutaneous lesion of a 6-year-old boy. This finding suggests that modifications of the skin lesion environment, such as maceration and changes in pH or temperature, could promote the in situ transformation of Leishmania amastigotes into promastigotes. This observation raises questions about the physiopathology of cutaneous leishmaniasis and the influence of micro-environmental changes on the efficiency of topical treatments.

First detection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Culicoides spp. (Diptera: Ceratopogonidae).

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) species are known to be the vectors of Bluetongue virus and African Horses Sickness virus (AHSV) in different areas of the world. Nevertheless, other researchers have hypothesized that these arthropods could be involved in the transmission of other pathogens such as Schmallenberg virus, Plasmodium and Leishmania parasites. Identification of the Culicoides' potential vector competence is crucial in understanding the worldwide Culicoides/Leishmania life cycle. FINDINGS: Blood fed and parous females of biting midges Culicoides spp. were collected between 2009 and 2010 in Central Tunisia. DNA was extracted from individual blood fed Culicoides and used as a template in a genus-specific PCR. Leishmania DNA was detected in 14 Culicoides imicola specimens and one Culicoides circumscriptus. In a second step, parasite identification was performed based on a single copy Topo-isomerase II gene specific amplification and sequencing. Leishmania infantum was identified in two infected Culicoides spp. CONCLUSION: This is the first report of Leishmania DNA detection from naturally infected wild caught Culicoides spp. Our finding supports the assumption that Culicoides spp. are a potential vector for L. infantum.