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.

Isoenzymatic variability of Leishmania infantum in Tunisia concerning 254 human strains.

The different clinical forms of leishmaniasis are the result of both the immunological status of individuals and the species of the parasite causing the infection. In Mediterranean countries, the Leishmania infantum complex groups zymodemes which are responsible for visceral, cutaneous and exceptionally cutaneomucosal or mucosal leishmaniasis. We report in this study a synthesis concerning 254 cases of L. infantum that have been characterized at the "Laboratoire de Parasitologie" of the Rabta Hospital. The strains were isolated from human cases of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) by culture on NNN medium: 156 VL cases and 98 CL cases. The isoenzymatic characterization revealed three zymodemes of L. infantum. * L. infantum MON 1, a common zymodeme of VL,occurred in 154 cases (61%): 147 VL (95%) and 7 CL (5%). All CL cases were from the northern provinces, six of them occurring during an epidemic disease in 2001. * L. infantum MON 24, a common zymodeme of CL in the north, occurred in 98 cases (38.5%): 91 CL (93%) and 7 VL (7%). The seven VL cases were immunocompetent children aged from 8 months to 9 years and native of northern Tunisia. Two of the CL cases were from central regions of the country. This is the first time that cases from these regions are reported. * L. infantum MON 80, an uncommon zymodeme in Tunisia, occurred in two VL cases (0.5%): two children aged 7 and 5. The small number of strains of this zymodeme does not allow understanding of its epidemiological role. The results of this study indicate a low enzymatic variability of L. infantum in the country. However, our study includes only human strains and should be extended to animal ones (dogs, rodents and sand flies). This would lead to a better understanding of the epidemiology of leishmaniasis in Tunisia.

[Cutaneous leishmaniasis caused by Leishmania infantum MON-24 in Tunisia: extension of the focus to the center of the country]. / La leishmaniose cutanée due a Leishmania infan- tum MON-24 en Tunisie: extension du foyer vers le centre du pays.

Three clinico-epidemiological forms of cutaneous leishmaniasis (CL) were described in Tunisia: the zoonotic CL (ZCL) epidemic which occurred in the centre of the country caused by Leishmania major MON-25, the chronic CL (CCL) In the south-east of the country caused by Leishmania killicki MON-8 and the sporadic CL In the North (SCL) caused by Leishmania infantum MON-24. The latter form, described in 1991, prevails in northern Tunisia with approximately thirty cases per year. Its vector, unknown for a long time could be according to the last publications, Phlebotomus perfiliewi or Phlebotomus langeroni; however, its reservoir remains unknown until now. The systematic isoenzymatic characterization permits to identify a great number of strains improving then knowledge on the eco-epidemiology of the disease. Indeed, changes were noted in the geographical distribution of these clinical forms: extension of the ZCL to the North and South, extension of the CCL to North and the SCL to the centre. We report in this note the first mention of L. infantum MON-24 in the two provinces of the centre of Tunisia: Kairouan and Sidi Bouzid, confirming the extension of the SCL to the Centre.

[Epidemiology of Leishmania (L.) infantum, L. major and L. killicki in Tunisia: results and analysis of the identification of 226 human and canine isolates]. / Epidémiologie de Leishmania (L.) infantum, L. major et L. killicki en Tunisie: résultats et analyse de l'identification de 226 isolats humains et canins et revue de la littérature.

The epidemiological situation of leishmaniasis in Tunisia is characterised by the co-existence in a very limited territory (165,000 km2, Sahara included), of 4 clinical forms: the infantile visceral leishmaniasis (VL) and 3 cutaneous leishmaniasis (CL) forms (sporadic, zoonotic and chronic). In addition to the useful epidemiological data, identification of the causative parasitic species is essential to determine the geographic distribution of each form and to select appropriate therapeutic procedure and suitable control measures. 226 Leishmania isolates, 135 human's coming from 59 VL cases and 76 CL cases and 91 canine's were identified by the isoenzyme electrophoresis reference technique. Results confirm the endemicity of the 4 forms mentioned above. The sporadic CL, confined to the North of the country is principally caused by L. infantum MON-24 (72.2%). VL which has reached the southern ridge in the central area of Tunisia, in the governorate of Kairouan (36 typed isolates), presents an unusual high proportion of L. infantum MON-24. In fact, this zymodeme, rather dermotropic is responsible for 47.2% of the cases vs 13% in the other regions of the country where L. infantum MON-1 remains predominant with 78.3% of typed isolates, the difference being statistically significant (P < 0.01). A third zymodeme, L. infantum MON-80 is sporadically pointed out during VL or in sporadic CL. Despite the high number of canine isolates (n=91) coming from 6 governorates, only the zymodeme L. infantum MON-1 was identified, letting hypothetic the reservoir of the 2 other zymodemes of the species identified in humans. Those absences may be related to cross infections, with a low sensitivity to L. infantum MON-24 leading to a selection of MON-1 at the time of culture passages. Hence it is important to develop molecular tools of direct identification on initial biological samples without going through cultures. Zoonotic CL remains the predominant cutaneous form in the central and southern area of Tunisia. However L. killicki, agent of the chronic CL, is confirming its presence out of its original focus of Tataouine in the southern-east of the country in both zoonotic CL and VL areas.

Increase of canine leishmaniasis in a previously low-endemicity area in Tunisia.

An epidemiological study of canine leishmaniasis (CanL) was carried out in nine districts of Sfax, in the southern central part of Tunisia. Sera from 250 dogs were tested by two serological methods: the indirect immunofluorescence antibody test and the counter-immunoelectrophoresis. Seven to eight months later, before the next season of transmission, seropositive dogs from the first test were re-examined and a second sampling was performed. Infection status was assessed by serology and by other methods. PCR, in vitro culture and direct examination were applied on blood and other samples (bone marrow, liver, lymph node, spleen and cutaneous biopsies). The seroprevalence of the infection in dogs was 6%. Infection was then confirmed by at least one other method. The PCR is the method which agreed most with serology, all seropositive dogs were found PCR-positive. The sensitivity of the direct examination and the culture was only 33% and 55% respectively as compared with serology. A similar value of seroprevalence has been observed previously in Sousse, in the northern central part of Tunisia. The present report suggests a significant increase of CanL in the Sfax area and confirms that the disease is continuing to move southwards in Tunisia.

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).

[Primary localized bladder amyloidosis]. / L'amylose vésicaoe primitive localisée.

Amyloidosis constitutes a group of storage diseases concerning various proteins, classified as a function of the type of protein. Localized pseudo tumoral amyloidosis of the urinary tract is a rare affection with bladder like the most frequent localisation. In spite of contribution of computerized tomography scans and MRI, the diagnosis is always based on histological examination of cystoscopy samples. Investigations are required to eliminate a generalized amyloidosis or a malignant lymphoproliferation. Treatment should be as conservative as possible on the outside of a massive and recurrent haematuria to be able to involve vital prognosis.

[Uretrovaginal reflux during micturition: A case report]. / Le reflux urétro-vaginal permictionnel.

Reflux into vagina during micturition is a rare phenomenon in adults. We report a case of a 22-old-year woman who presented with intravaginal influx of urine. This case illustrates the anatomic basis, diagnostic examinations and clinical and bacteriological implications of this phenomenon.

[Intravesical migration of intrauterine device resulting in stone formation]. / Migration intra-vésicale de dispositif intra-utérin compliquée de lithiase.

The migration into the bladder of an intrauterine contraceptive device (IUCD) by uterine perforation is a rare complication. We report two cases of IUCD which migrated into the bladder and subsequently became calcified. The two patients having had their IUCD respectively for 3 and 13 years. Revealing signs were related to bladder irritation for the first patient and hematuria for the second. The diagnosis was suggested on the plain abdominal X-ray and on ultrasound and was confirmed by cystoscopy. Ballistic lithotripsy of the bladder stone with endoscopic extraction of the IUCD was then performed. Performing a transvaginal sonographic examination of the pelvic organs, especially of the uterine anatomy is interesting before insertion of an intrauterine contraceptive device (IUCD), and repeat transvaginal sonographic examinations immediately after the insertion and 4-12 weeks later are advisable. This approach would permit early detection of any complications related to insertion of the IUCD.

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.

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.

[Epidemiological, clinical and parasitological data about canine leishmaniasis in Tunisia]. / Données epidémiologiques, cliniques et parasitologiques sur la leishmaniose générale canine en Tunisie.

Epidemiological, clinical and parasitological data concerning canine leishmaniasis were collected in two Tunisian populations of dogs, different in breed and life style: 23 rural dogs and 26 dogs of European breeds. All were symptomatic and lived in the north of Tunisia where human visceral and cutaneous leishmaniasis caused by Leishmania infantum are endemic. Leishmaniasis has been confirmed in all dogs by serology or/and by parasitic identification. Significant differences concerning age and symptoms suggest a higher susceptibility to infection in European imported breeds. In fact individuals of this population were significantly younger; 81% were less than 5 years old whereas 57% of the autochthonous rural dogs were more than 5 years old with 31% of them being older than 9 (p = 0.014). In spite of their young age, 75% of imported breeds presented an affection of their general state with more frequent cutaneous symptoms than the rural dogs (96% versus 69%, p = 0.02). Isoenzyme typing of 31 strains, obtained from the two populations, from different sites (blood, lymph nodes, spleen) has only identified the zymodeme Leishmania infantum MON-1. This stresses the need of more investigations to determine reservoirs of the other enzymatic variants identified in humans in Tunisia and Mediterranean basin.

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.