Malaria screening: what is the contribution of molecular biology?

INTRODUCTION: According to the World Health Organization, Microscopy is the gold standard for diagnosing malaria. However, the performance of this examination depends on the experience of the microscopist and the level of parasitemia. Thus, molecular biology detection of malaria could be an alternative technique. AIM: evaluate the contribution of molecular biology in detecting imported malaria. METHODS: This was a descriptive, prospective study, including all students, from the Monastir region, and foreigners, from countries endemic to malaria. The study period was from September 2020 to April 2021. Each subject was screened for malaria by three methods: direct microscopic detection of Plasmodium, detection of plasmodial antigens, and detection of plasmodial DNA by nested PCR. RESULTS: Among the 127 subjects screened, only one had a positive microscopic examination for Plasmodium falciparum. Among the 126 subjects with a negative microscopic examination, twelve students had a positive nested PCR result, i.e. 9.5%. Molecular sequencing allowed the identification of ten isolates of Plasmodium falciparum, one Plasmodium malariae and one Plasmodium ovale. Our study showed that the results of nested PCR agreed with those of microscopy in 90.6% of cases. CONCLUSION: Nested PCR seems more sensitive for the detection of low parasitemias. Hence the importance of including molecular biology as a malaria screening tool to ensure better detection of imported cases.

Synthesis, Characterization, X-ray Molecular Structure, Antioxidant, Antifungal, and Allelopathic Activity of a New Isonicotinate-Derived meso-Tetraarylporphyrin.

The present article describes the synthesis of an isonicotinate-derived meso-arylporphyrin, that has been fully characterized by spectroscopic methods (including fluorescence spectroscopy), as well as elemental analysis and HR-MS. The structure of an n-hexane monosolvate has been determined by single-crystal X-ray diffraction analysis. The radical scavenging activity of this new porphyrin against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical has been measured. Its antifungal activity against three yeast strains (C. albicans ATCC 90028, C. glabrata ATCC 64677, and C. tropicalis ATCC 64677) has been tested using the disk diffusion and microdilution methods. Whereas the measured antioxidant activity was low, the porphyrin showed moderate but encouraging antifungal activity. Finally, a study of its effect on the germination of lentil seeds revealed interesting allelopathic properties.

Identification and antifungal susceptibility profile of uncommon yeast species at Fattouma Bourguiba University Hospital in Tunisia.

Despite the severe impact of uncommon yeast fungal infections and the pressing need for more research on the topic, there are still few studies available on the identification, epidemiology, and susceptibility profile of those pathogens. The aims of the current study were to define the profile of uncommon yeast species at Fattouma Bourguiba University Hospital using phenotypic, molecular, and proteomic methods and to study their antifungal susceptibility profile. Pre-identified uncommon yeast species were collected from 2018 to 2021. These isolates were further identified using phenotypic methods (ID32C® system and Vitek2® YST), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and sequencing. The antifungal susceptibility profile was studied using the reference CLSI broth microdilution method. In total, 30 strains were collected during the study period. Referring to the sequencing, the most isolated uncommon species were Saprochaete capitata, Candida lusitaniae, Candida kefyr, Candida inconspicua, and Candida guilliermondii. A total of 90% of isolates were correctly identified by MALDI-TOF MS compared to 76.7% and 63.3% by ID32® C and VITEK® 2 YST, respectively. The isolated species showed variable responses to antifungals. Candida guilliermondii showed increased azole minimum inhibitory concentrations. Misidentification of uncommon yeast species was common using commercial phenotypic methods. The high percentage of concordance of MALDI-TOF results with sequencing highlights its high performance and usefulness as a routine diagnosis tool.

There is still little information on the epidemiology of uncommon emergent yeasts, although their implication in severe diseases and mainly invasive infections. Thus, the importance of an accurate identification and antifungal susceptibility testing for a better monitoring of related infections.

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.

Chemical composition, antifungal and antibiofilm activities of essential oils from Glycyrrhiza foetida (Desf.) growing in Tunisia.

This study was designated to investigate the chemical composition, the antifungal activity and antibiofilm properties of Glycyrrhiza foetida (Desf.) growing in Tunisia and recognized for its pharmacological and therapeutic effects. The chemical analysis of essential oil samples prepared via hydrodistillation of the aerial parts was performed by gas chromatography-mass spectrometry (GC-MS). Moreover, the antifungal activity of G. foetida essential oil was developed against three dermatophyte strains, two molds and Candida spp. yeasts using the broth microdilution assay. According to the percentages, the main constituents are δ-cadinene (13.9%), (E)-caryophyllene (13.2%) and γ-cadinene (8.3%). The efficiency of the essential oil in inhibiting Candida albicans biofilms formation was also evaluated in terms of inhibitory percentages. The results showed that C. albicans and Microsporum canis were the most sensitive to G. foetida essential oil with a complete inhibition at 0.4 and 0.2 mg ml-1 , respectively. Candida albicans biofilm development was reduced by 80% by the volatile oil at a concentration of 0.8 mg ml-1 . The essential oil of G. foetida has a promising role in the control of fungal agents with medical interest and in inhibition of Candida biofilm development.

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.

Chemical Composition, Antifungal and Anti-Biofilm Activities of Volatile Fractions of Convolvulus althaeoides L. Roots from Tunisia.

The antifungal drugs currently available and mostly used for the treatment of candidiasis exhibit the phenomena of toxicity and increasing resistance. In this context, plant materials might represent promising sources of antifungal agents. The aim of this study is to evaluate for the first time the chemical content of the volatile fractions (VFs) along with the antifungal and anti-biofilm of Convolvulus althaeoides L. roots. The chemical composition was determined by gas chromatography coupled to a flame ionization detector and mass spectrometry. In total, 73 and 86 chemical compounds were detected in the n-hexane (VF1) and chloroform (VF2) fractions, respectively. Analysis revealed the presence of four main compounds: n-hexadecenoic acid (29.77%), 4-vinyl guaiacol (12.2%), bis(2-ethylhexyl)-adipate (9.69%) and eicosane (3.98%) in the VF extracted by hexane (VF1). n-hexadecenoic acid (34.04%), benzyl alcohol (7.86%) and linoleic acid (7.30%) were the main compounds found in the VF extracted with chloroform (VF2). The antifungal minimum inhibitory concentrations (MICs) of the obtained fractions against Candida albicans, Candida glabrata and Candida tropicalis were determined by the micro-dilution technique and values against Candida spp. ranged from 0.87 to 3.5 mg/mL. The biofilm inhibitory concentrations (IBF) and sustained inhibition (BSI) assays on C. albicans, C. glabrata and C. tropicalis were also investigated. The VFs inhibited biofilm formation up to 0.87 mg/mL for C. albicans, up to 1.75 mg/mL against C. glabrata and up to 0.87 mg/mL against C. tropicalis. The obtained results highlighted the synergistic mechanism of the detected molecules in the prevention of candidosic biofilm formation.

In Vitro Assessment of Azole and Amphotericin B Susceptibilities of Malassezia spp. Isolated from Healthy and Lesioned Skin.

Malassezia yeasts have recently gained medical importance as emerging pathogens associated with a wide range of dermatological and systemic infections. Since standardized methods for in vitro antifungal susceptibility testing have not yet been established for Malassezia spp., related diseases are always treated empirically. As a result, a high rate of recurrence and decreased antifungal susceptibility have appeared. Thus, the aims of the study were to assess and analyze the in vitro susceptibility of Malassezia isolated from pityriasis versicolor (PV) lesions and healthy controls. A total of 58 Malassezia strains isolated from PV patients and healthy controls were tested. In vitro antifungal susceptibility testing was conducted using the CLSI broth microdilution with some modifications. Candida spp. criteria established in accordance with CLSI guidelines were used for data interpretation. Ketoconazole and posaconazole seemed to be the most effective molecules against Malassezia species. However, considerable percentages of itraconazole, fluconazole, and amphotericin B ''resistant'' strains (27.6%, 29.3%, and 43.1%, respectively) were revealed in this study. Malassezia furfur, M. sympodialis, and M. globosa showed different susceptibility profiles to the drugs tested. These results emphasize the importance of accurately identifying and evaluating the antifungal susceptibility of Malassezia species in order to guide a specific and effective treatment regimen.

Virulence factors of Malassezia strains isolated from pityriasis versicolor patients and healthy individuals.

Over the last decade, Malassezia species have emerged as increasingly important pathogens associated with a wide range of dermatological disorders and bloodstream infections. The pathogenesis of Malassezia yeasts is not completely clear, but it seems to be strictly related to Malassezia strains and hosts and needs to be better investigated. This study aimed to assess the enzymatic activities, biofilm formation and in vitro antifungal profiles of Malassezia spp. from pityriasis versicolor (PV) and healthy patients. The potential relationship between virulence attributes, the antifungal profiles and the origin of strains was also assessed. A total of 44 Malassezia strains isolated from patients with (n = 31) and without (n = 13) PV were employed to evaluate phospholipase (Pz), lipase (Lz), and hemolytic (Hz) activities and biofilm formation. In addition, in vitro antifungal susceptibility testing was conducted using the CLSI broth microdilution with some modifications. A high percentage of strains produced Pz, Lz, Hz and biofilm regardless of their clinical origin. The highest number of strains producing high enzymatic activities came from PV patients. A correlation between the intensity of hydrolytic activities (Lz and Pz activities) and the Hz activity was detected. Positive associations between Lz and the low fluconazole susceptibility and Hz and biofilm formation were observed. These results suggest that enzyme patterns and biofilm formation along with antifungal profiles inter-play a role in the pathogenicity of Malassezia spp. and might explain the implication of some Malassezia spp. in invasive fungal infections and in the development of inflammation. LAY SUMMARY: There is still little information on the virulence factors of Malassezia spp., despite their implication in severe diseases. Phospholipase, lipase, and hemolytic activities, biofilm formation and decreased antifungal susceptibility seem to contribute to their virulence in susceptible hosts.

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.

Comparison of virulence factors and susceptibility profiles of Malassezia furfur from pityriasis versicolor patients and bloodstream infections of preterm infants.

In spite of the increasing medical interest in Malassezia yeasts, the virulence factors of Malassezia furfur causing bloodstream infections (BSI) were never investigated. Therefore, phospholipase (Pz), lipase (Lz), hemolysin (Hz), biofilm production, and in vitro antifungal susceptibility profiles were evaluated in M. furfur strains, isolated from both pityriasis versicolor (PV) patients (n = 18; Group 1) or from preterm infants BSI (n = 21; Group 2). All the test stains exhibited Pz activity, whereas 92.3% and 97.4% of strains exhibited Lz and Hz activities, respectively. Pz, Lz, and Hz activities were higher (i.e., lower values) within Group 1 strains (i.e., 0.48, 0.40, and 0.77) than those within Group 2 (i.e., 0.54, 0.54, and 0.81). The biofilm production was higher within Malassezia isolates from Group 2 (0.95 ± 0.3) than from Group 1 (0.72 ± 0.4). Itraconazole and posaconazole were the most active drugs against M. furfur, followed by amphotericin B and fluconazole. The minimum inhibitory concentrations (MIC) values varied according to the origin of M. furfur strains being statistically lower in M. furfur from Group 1 than from Group 2. This study suggests that M. furfur strains produce hydrolytic enzymes and biofilm when causing PV and BSI. Data show that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur BSI, whereas lipase and hemolytic activities might display a synergic role in skin infection.

There is no information on the virulence factors of M. furfur involved in invasive infections. Our data suggest that the phospholipase activity, biofilm production, and a reduced antifungal susceptibility profile might favor M. furfur blood-stream infections.

Novel biomarkers for the early prediction of pediatric cystic echinococcosis post-surgical outcomes.

OBJECTIVE: This study aims to search for reliable serological biomarkers allowing the early prediction of cystic echinococcosis (CE) post-operative outcomes. METHODS: We applied immunoprecipitation (IP) of Echinococcus granulosus protoscolex antigens with pediatric CE patients' plasma collected at 1-month and 1-year post-surgery, followed by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). We compared IP proteomic content from relapsed patients within the first-year post-surgery (RCE) to cases with no relapses until 3 post-operative years (NRCE). Selected proteins were recombinantly synthesized and assessed for their prognostic performance by Enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 305 immunoreactive parasitic proteins were identified, 59 of which were significantly more abundant in RCE than NRCE for both time-points. Four proteins showed the most promising characteristics for predicting CE outcomes: cytoplasmic malate dehydrogenase (Eg-cMDH), citrate synthase (Eg-CS), annexin A6 and severin. ELISA-IgG against the four markers were significantly lower at 1-year post-surgery than 1-month in NRCE, in contrast to RCE that displayed either stable or higher levels. The Eg-cMDH and Eg-CS showed the best prognostic performance, with respective probabilities of being "relapse-free" of 83% and 81%, if a decrease of IgG levels occurred between 1-month and 1-year post-surgery. CONCLUSION: The Eg-cMDH and Eg-CS are promising biomarkers to predict early CE post-surgical outcomes.

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.

First isolation and genotyping of Toxoplasma gondii strains from domestic animals in Tunisia.

The isolation and molecular typing of Toxoplasma gondii strains provide an essential basis for a better understanding of the parasite's genetic diversity, determinants of its geographical distribution and associated risks to human health. In this study, we isolated and genetically characterized T. gondii strains from domestic animals in Southern and coastal area of Tunisia. Blood, hearts and/or brains were collected from 766 domestic animals (630 sheep and 136 free-range chickens). Strain isolation from these samples was performed using mouse bioassay and genotyping was carried out with a multiplex PCR technique using 15 microsatellite markers. Thirty viable strains of T. gondii were successfully isolated from tissues of sheep (19/142) and chickens (11/33). In addition, 3 strains could be successfully genotyped from animal tissues for which mouse bioassay was unsuccessful. A large predominance of type II strains (n = 29) was found in the sampled regions, followed by type III (n = 3) and, for the first time in Tunisia, a single isolate of Africa 4 lineage from a sheep. Analyses of population genetics showed the presence of a divergent population of type II lineage in Tunisia, supporting limited recent migrations of strains between Tunisia and other countries of the world.

Human and animal cystic echinococcosis in Tataouine governorate: hypoendemic area in a hyperendemic country, myth or reality?

BACKGROUND: Cystic echinococcosis (CE) has a worldwide distribution and is especially prevalent in North African countries. With a mean annual surgical incidence (ASI) of CE of 12.7 per 100,000 inhabitants, Tunisia is one of the most CE endemic countries in the Mediterranean area. Tataouine governorate is considered to be the most CE hypoendemic region in Tunisia (ASI = 0.92) despite favourable socioeconomic conditions that enable maintenance of the Echinococcus granulosus sensu lato (s.l.) life-cycle and a significant environmental contamination with E. granulosus s.l. eggs. The aim of this study was to assess human CE seroprevalence, prevalence of CE in food animals and environmental contamination by E. granulosus s.l. eggs in different districts of Tataouine governorate. METHODS: This study was conducted from January to December 2018. A total of 374 human sera samples were tested for the presence of immunoglobulin G antibodies against E. granulosus using a commercial ELISA kit. Specimens were also collected from animals slaughtered at the Tataouine abattoir (n = 8609) and examined for the presence of hydatid cysts; 111 hydatid cysts were genotyped. Eggs of E. granulosus s.l. were identified by PCR and DNA sequencing from dog faecal samples (n = 288). RESULTS: Serological tests showed that 8.5% of the sera samples tested were positive for E. granulosus-specific antibodies. The average prevalence of hydatidosis in livestock was 1.6%, and CE infection was more prevalent in cattle than in sheep, goats and dromedaries. The contamination rate of dog faeces by E. granulosus sensu stricto eggs varied significantly from 0 to 23.5% depending on the collection area. Molecular analyses only revealed the presence of the G1 genotype for cysts and eggs. CONCLUSIONS: Based on our findings, CE is likely to be more endemic in the Tataouine governorate than previously described. Thus, to implement an effective control programme against CE, a national survey should be carried out to determine human CE prevalence in the different Tunisian governorates.

Promising proteins detected by Western blot from Echinococcus granulosus protoscoleces for predicting early post-surgical outcomes in CE-affected Tunisian children.

BACKGROUND: Cystic echinococcosis (CE) affects predominantly young patients in highly endemic areas. Improved serological methods are needed for the follow-up of CE cases, especially given the high rates of post-surgical relapse that require detection as soon as possible. METHODS: We designed a study to investigate the value of antigenic proteins extracted from Echinococcus granulosus (E. granulosus) protoscoleces, and of recombinant B2t and 2B2t proteins, for assessing the efficacy of surgical treatment carried out on CE-affected children. This study was performed on 278 plasma samples collected from 59 Tunisian children surgically treated for CE and monitored for 3 years post-surgery. The patients were classified according to post-surgical outcomes into a "non-relapsed" (NRCE) and a "relapsed" (RCE) group. We performed in-house ELISAs to measure anti-B2t and anti-2B2t IgG and immunoblotting for the detection of IgG against SDS-PAGE-resolved E. granulosus protoscoleces-specific antigens. The Wilcoxon test was applied to assess anti-B2t and anti-2B2t IgG levels. We applied the Cochran Q test to compare the distribution of immunoblotting antigenic bands between 1-month and 1-year post-surgery. RESULTS: The probability of being "relapse-free" when a decrease in antibody titers occurred between 1 month and 1 year post-surgery was 81% and 75%, respectively, for anti-B2t and anti-2B2t IgG. We identified five protoscolex protein bands of 20, 26/27, 30, 40 and 46 kDa as highly immunoreactive by immunoblot for both RCE and NRCE patients at 1 month post-surgery, and significantly lower immunoreactivity after 1 year (p < 10-4) for NRCE compared to RCE patients. The proteins at 26/27 and 40 kDa displayed the best performance in predicting the outcome, with an 84% probability of being relapse-free when the reactivity against the 40 kDa antigen, the doublet at 26/27 kDa, or both was absent or disappeared between 1 month and 1 year post-surgery, and a 93% probability of being relapsed when both bands remained reactive or increased in intensity between the two time points. CONCLUSIONS: The B2t protein could be useful for the prediction of CE early post-surgical outcomes. The proteins of E. granulosus protoscoleces, especially the doublet P26/27 and P40, could be promising predictive biomarkers for the post-surgical follow-up of CE cases as well.

Soluble programmed death-1 (sPD-1) as predictor of early surgical outcomes of paediatric cystic echinococcosis.

AIMS: Following treatment, cystic echinococcosis (CE) exhibits a relatively high relapse rate. Here, we evaluated the value of soluble programmed death-1 (sPD-1), sPD-1 ligand (sPD-L1) and anti-recP29 antibody concentrations, as predictors of early surgical treatment outcomes in young CE-affected patients. METHODS AND RESULTS: This prospective study included 59 Tunisian children (177 plasmas), where CE was surgically treated and monitored for 3 post-operative years. Based on CE post-surgical development, patients were clustered into a 'No relapsed' CE (NRCE; n = 39) and a 'Relapsed' CE (RCE; n = 20) group. Plasma levels of sPD-1, sPD-L1 and anti-recP29 IgG were measured using ELISA. In the NRCE group, sPD-1, sPD-L1 and anti-recP29 IgG concentrations were significantly lower at D365 than at D30. By contrast, in the RCE group, no significant difference was observed between D0, D30 and D365. When considering individual variations, the probability to be 'relapse-free' was 67% and 73% when anti-recP29 IgG and sPD-L1 level, respectively, decreased between D30 and D365. The probability to be 'relapse-free' was 86% when the sPD-1 level decreased between D30 and D365 (P = .003; chi-square test). CONCLUSION: sPD-1 may be a useful biomaker for the early evaluation of surgical procedure efficacy in paediatric CE cases.

Botanical and Genetic Identification Followed by Investigation of Chemical Composition and Biological Activities on the Scabiosa atropurpurea L. Stem from Tunisian Flora.

Scarce information about the phenolic composition of Scabiosa atropurpurea L. is available, and no carotenoid compounds have been reported thus far. In this study the phenolic and carotenoid composition of this plant was both investigated and associated bioactivities were evaluated. Aiming to obtain extracts and volatile fractions of known medicinal plants to valorize them in the pharmaceutical or food industries, two techniques of extraction and five solvents were used to determine the biologically active compounds. Gas chromatography coupled to flame ionization and mass spectrometry and liquid chromatography coupled to photodiode array and atmospheric pressure chemical ionization/electrospray ionization mass spectrometry highlighted the presence of 15 volatiles, 19 phenolic, and 24 natural pigments in Scabiosa atropurpurea L. stem samples; among them, the most abundant were 1,8-cineole, chlorogenic acid, cynaroside, and lutein. Bioactivity was assessed by a set of in vitro tests checking for antioxidant, antibacterial, antifungal, and allelopathic (against Brassica oleracea L. and Lens culinaris Medik) effects. Scabiosa atropurpurea L. stem extracts presented a considerable antioxidant, antibacterial, and allelopathic potential, with less antifungal effectiveness. These results indicate that the volatile fractions and extracts from S. atropurpurea L. stem could be considered as a good source of bioactive agents, with possible applications in food-related, agriculture, and pharmaceutical fields. Genetic investigations showed 97% of similarity with Scabiosa tschiliensis, also called Japanese Scabiosa.