Presence of antimicrobial resistance in coliform bacteria from hatching broiler eggs with emphasis on ESBL/AmpC-producing bacteria.

Antimicrobial resistance is recognized as one of the most important global health challenges. Broilers are an important reservoir of antimicrobial resistant bacteria in general and, more particularly, extended-spectrum ß-lactamases (ESBL)/AmpC-producing Enterobacteriaceae. Since contamination of 1-day-old chicks is a potential risk factor for the introduction of antimicrobial resistant Enterobacteriaceae in the broiler production chain, the presence of antimicrobial resistant coliform bacteria in broiler hatching eggs was explored in the present study. Samples from 186 hatching eggs, collected from 11 broiler breeder farms, were inoculated on MacConkey agar with or without ceftiofur and investigated for the presence of antimicrobial resistant lactose-positive Enterobacteriaceae, particularly, ESBL/AmpC-producers. Escherichia coli and Enterobacter cloacae were obtained from the eggshells in 10 out of 11 (10/11) sampled farms. The majority of the isolates were recovered from crushed eggshells after external decontamination suggesting that these bacteria are concealed from the disinfectants in the egg shell pores. Antimicrobial resistance testing revealed that approximately 30% of the isolates showed resistance to ampicillin, tetracycline, trimethoprim and sulphonamides, while the majority of isolates were susceptible to amoxicillin-clavulanic acid, nitrofurantoin, aminoglycosides, florfenicol, neomycin and apramycin. Resistance to extended-spectrum cephalosporins was detected in eight Enterobacteriaceae isolates from five different broiler breeder farms. The ESBL phenotype was confirmed by the double disk synergy test and blaSHV-12, blaTEM-52 and blaACT-39 resistance genes were detected by PCR. This report is the first to present broiler hatching eggs as carriers and a potential source of ESBL/AmpC-producing Enterobacteriaceae for broiler chicks.

Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

Geographical distribution updating of Tunisian leishmaniasis foci: about the isoenzymatic analysis of 694 strains.

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.

[Retrospective study of the distribution and the fertility of hydatid cysts in the child in Tunisia]. / Étude rétrospective de la distribution et de la fertilité des kystes hydatiques chez l'enfant en Tunisie.

PURPOSE OF THE STUDY: Our aim was to study the distribution and the fertility of the hydatid cysts in function of the age and the sex of patients and to identify the strain(s) responsible(s) of the children hydatidosis. PATIENTS AND METHODS: We have analyzed a total of 241 cysts coming from 195 children aged 2 to 16 years operated in the CHU F. Bourguiba of Monastir during the period from November 1999 to December 2009. For each cyst, the localization and the fertility of the métacestode as well as age, sex and origin of the patient are listed. Identification of strains was carried out by PCR/RFLP and has targeted the ribosomal gene ITS1. RESULTS: The lung was the primary localization of cyst (61.8%) followed by the liver (34.85%). The greatest number of cases is observed in the age groups 4-9 years (138 cases) where children's infection is more frequent in the male than in the female sex. The fertility of the cyst was independent of its site or its size and no incidence of age of children was detected. The G1 sheep strain is responsible for the contamination of children. CONCLUSION: The cystic echinococcosis described as a young adult disease may actually observed at any age and remains a serious problem of public health in Tunisia.

Usefulness of a PCR-based method in the detection and species identification of Leishmania from clinical samples.

The aim of this study is to assess the usefulness of a simple, low-cost method for the detection and species identification of Leishmania isolated by in vitro culture or detected directly from clinical samples. A total of 110 samples were used in this study. Among these, 21 were human and canine peripheral bloods, 63 skin lesion material samples, eight reference strains and 18 Leishmania culture. Detection of Leishmania DNA with PCR using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently sensitive at the level of 0.1 parasites per PCR reaction. Furthermore, followed by single-strand conformational polymorphism (SSCP), the PCR-ITS1 allowed the species identification of Leishmania. The inter-specific polymorphism of Leishmania was first validated on reference strains, and then this method was applied on clinical samples and culture. Typing identified all human and canine visceral leishmaniasis samples (21 samples) as L. infantum, 95.23% of the cutaneous leishmaniasis samples as L. major and 3.17% as L. killicki and 1.58% as L. infantum. A scheme of the PCR diagnosis procedure for the detection and identification of Leishmania parasites is proposed in this study.

Use of PCR, IFAT and in vitro culture in the detection of Leishmania infantum infection in dogs and evaluation of the prevalence of canine leishmaniasis in a low endemic area in Tunisia.

The aim of this study was to assess the use of parasitological, serological and molecular methods for the detection of Leishmania infection in blood of 67 dogs and to investigate the prevalence of canine leishmaniasis (CanL) in Kairouan (central Tunisia), an area known to be of reduced endemicity and has not been studied since 1973. Veterinarians clinically examined all dogs, and the titer of anti-Leishmania antibodies was determined by indirect immune-fluorescence antibody test. The presence of Leishmania was performed by PCR and in vitro culture. IFAT was positive in 12% of dogs and promastigote form of the parasite was isolated by in vitro culture from only 4.5% of them. However, DNA of Leishmania was detected by PCR in 20.9% of dogs. PCR was more sensitive than IFAT (p = 0.004) and in vitro culture (p < 10(-5)). A prevalence of 21% was found in Kairouan, which is significant high (p < 10(-3)) when compared to that of thirty years ago. This state is in correlation with the increase in other Mediterranean countries. Furthermore, 50% of positive dogs were asymptomatic. Preventive measures must be taken against these dogs as for symptomatic ones since their role in the transmission of the infection to vectors has been proven.

The phlebotomine fauna (Diptera: Psychodidae) of the eastern coast of Tunisia.

To identify the phlebotomine sand fly populations of the eastern coast of Tunisia, an entomological survey was carried out between September and October 2005 at 71 sites located in three districts. CDC light traps and sticky papers were used to collect a total of 2,138 phlebotomine sand flies representing nine species. The predominant species occurring on the eastern coast of Tunisia are, in order of abundance, Phlebotomus longicuspis Nitzulescu, 1930 (40%); Phlebotomus papatasi Scopoli, 1786 (21%); Sergentomyia minuta Parroti Adler & Theodor, 1927 (19%); Phlebotomus perniciosus Newstead, 1911 (9.5%); Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 (9%); Sergentomyia fallax Parrot, 1921 (1.5%); Sergentomyia dreyfussi Parrot, 1933 (0.23%); Phlebotomus langeroni Nitzulescu, 1930 (0.05%); and Phlebotomus perfiliewi Parrot, 1930 (0.05%). Species involved in the transmission of Leishmania, namely P. papatasi and P. perniciosus, represent 31% of the total number of flies captured. In the central sites (district of Monastir), P. longicuspis predominates, P. perniciosus predominates in the northern sites (district of Sousse) and P. papatasi in the southern sites (district of Mahdia), which is consistent with the distribution of Leishmania infantum and L. major in this region. Analysis of the degree of presence (D) revealed that Phlebotomus papatasi was the most common species and showed the broadest distribution (D = 95%), followed by P. longicuspis and S. minuta parroti (D = 90%) and P. perniciosus and P. chabaudi (D = 86% and 68% respectively).

Leishmaniasis in central and southern Tunisia: current geographical distribution of zymodemes.

The authors report the identification of Leishmania strains isolated from the Centre and the South of Tunisia. 266 strains were isolated between 1998 and 2006 from human (n=221 strains) and dogs (n=45 strains) hosts. The isoenzymatic identification exhibits the presence of in total five zymodemes belonging to three Leishmanio complexes: Leishmania infantum, L. major and L. killicki. All strains isolated from human and canine visceral leishmaniasis belonged to L. infantum. zymodeme MON-1 was the only one isolated from canine visceral leishmaniasis. However, it is predominant in human visceral leishmaniasis beside zymodeme MON-24 which was detected in two provinces of the Centre (Monastir and Kairouan) and zymodeme MON-80 isolated for the first time in Kairouan province. Three complexes are responsible for human cutaneous leishmaniasis: L. major MON-25 is the parasite the most frequently found in its classic foci in the Centre and the South of the country. L. infantum MON-24 was isolated for the first time in a small locality of Sfax (southern Tunisia) showing the appearance of a new focus of L. infantum. L. killicki was isolated in its original focus of Tataouine and in two new foci of the central part of the country (Sidi Bouzid and Kairouan).

[Cystic echinococcosis in children in Tunisia: fertility and case distribution of hydatid cysts]. / L'échinococcose hydatique de l'enfant en Tunisie: fertilité et localisation des kystes.

Cystic echinococcosis, which commonly starts during childhood or adolescence, is a serious problem of public health in Tunisia. For 121 children (161 cysts), the localization and fertility of cysts as well as viability of their protoscoleces were determined. Results indicated that the lung was the primary localization of cyst (59%) followed by the liver (35%). Children's infection is more frequent in male than in female (sex ratio 1.96) and the greatest number of cases is observed in the 4-9 year age groups (94 cases). The fertility of the cyst was independent of its site or its size and no incidence of age of children was detected. Nevertheless, the fertility rate is higher in females than in males for the liver localization.

Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP.

Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP) was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst= 0.46). No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing) was found in a limited number of camel isolates.

Usefulness of PCR in the diagnosis of cutaneous leishmaniasis in Tunisia.

We assessed the efficiency of a PCR method in establishing the diagnosis of cutaneous leishmaniasis (CL) in Tunisian patients. Four hundred and thirty specimens collected passively from patients with cutaneous ulcers suggestive of leishmaniasis attending health centres for diagnosis were included in the study. Dermal scrapings were analysed both by parasitological (examination of Giemsa-stained smears and in vitro cultivation) methods and by a genus-specific PCR detecting a fragment of the 18S rRNA gene. Microscopy revealed amastigotes in 245 samples (57.0%) and in vitro cultivation gave positive results in 88 cases (20.5%), whereas PCR detected Leishmania in 301 samples (70%). The sensitivities inferred from our results were 99.3%, 80.8% and 29% for PCR, microscopic examination and in vitro cultivation, respectively. The different forms of CL in this country are caused by three species of Leishmania and are treated with the same protocol. Of 303 well-documented cases in our study, 99% were probably caused by Leishmania major and 1% by Leishmania infantum. The lack of species-specific diagnosis is not known to affect treatment or prognosis in Tunisia. These data support the incorporation of PCR into diagnostic strategies for CL, particularly in Tunisia.

Anthroponotic cutaneous leishmaniasis in Tunisia: presence of Leishmania killicki outside its original focus of Tataouine.

The first three documented cases of anthroponotic cutaneous leishmaniasis due to Leishmania killicki are reported from locations outside the original focus of Tataouine in southeast Tunisia. Three strains were isolated from three patients from Gafsa, Sidi Bouzid and Seliana indicating an extension of this parasite's range towards the centre and the north of Tunisia.

Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination.

Three hundred and seventy-two cysts coming from 50 humans, 166 cattle, 153 sheep and 3 camels were collected in order to establish some epidemiological molecular information in Tunisia for the first time. The analysis by PCR-RFLP of ITS1 sequence showed that all the human, ovine and bovine cysts were due to the common sheep strain of Echinococcus granulosus. The sequencing of the CO1 gene of 37 isolates confirm the G1 genotype of this strain. For seven of these isolates, we found the mutation C56T which is present in the three principal intermediate hosts: human (three cysts), cattle (three cysts) and sheep (one cyst). With regard to the G1 genotype, we identified three other point mutations. The camel strain G6 is uniquely found in the three camels isolates and not in the other intermediate hosts analysed. The fertility of the bovine cyst represents 48% that means that this host is involved in a bovine-dog cycle and consequently represents a reservoir of sheep strain in Tunisia. Our results confirm the importance of the prophylaxis measures in order to disrupt the cycle of transmission sheep-dog in Tunisia. Nevertheless, the supervision of bovine infection should be reinforced because this intermediate host may constitute an important link with the human contamination.

Genetic relationships between sheep, cattle and human Echinococcus infection in Tunisia.

Allozyme variation at seven polymorphic loci (GPI, EST, MDH, MPI, DIA, PEP, PGM) was studied to examine genetic variation within and between sheep, cattle and human populations of Echinococcus granulosus in Tunisia. A high degree of genetic similarity was shown between the cysts of the three host origins. Nevertheless, whereas, the ovine and human samples were highly similar, the cattle samples were slightly different genetically. We conclude that humans are mostly infected by parasites originating from sheep liver. The intense deficiency in heterozygotes was partly artefactual (Wahlund effect) and partly due to self-fertilisation.