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.

Increase in the prevalence of canine leishmaniasis in urban Algiers (Algeria) following the 2003 earthquake.

Between 2005 and 2008, a serological survey for leishmanial infection was conducted among dogs from urban and peri-urban Algiers, with the focus on the new, densely populated areas that were built after the 2003 earthquake. Serum samples were collected from 1810 animals and tested for the presence of leishmanial antibodies by IFAT, ELISA and western blotting. The overall seroprevalence recorded was 25.1%. Of the seropositive dogs, 58.8% showed no clinical signs of the disease, 25.8% had a few, minor signs and the remaining 15.4% showed more severe illness. The major clinical signs of infection were weight loss, skin lesions and lymphadenopathy. Although seropositive dogs were found in all of the boroughs (daïras) of Algiers, seroprevalences were highest in the western part of the city (i.e. in the boroughs of Bouzaréah, Chéraga and Zéralda), ranging from 23.0% to 44.5%. Statistical analysis showed a relationship between seropositivity for leishmanial infection and the dog's age and lifestyle (i.e. whether the dog lived outside and/or in areas with dense vegetation). Only two zymodemes were identified amongst the 50 isolates investigated: MON-1 (88%) and MON-281 (12%). The latter zymodeme has not been previously found in Algeria, sandflies or dogs.

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.

First detection and genetic typing of Leishmania infantum MON-24 in a dog from the Moroccan Mediterranean coast: genetic diversity of MON-24.

As in the countries edging the Mediterranean basin, Leishmania infantum zymodeme MON-1 is the main causative agent of visceral leishmaniasis in Morocco, where visceral leishmaniasis is most active in the North-Eastern slopes of the Rif mountains. The dog was confirmed to be the main reservoir of L. infantum MON-1, while the reservoir of L. infantum MON-24 causative agent of both infantile visceral leishmaniasis and cutaneous leishmaniasis has not yet been identified. Here we report the first detection of this last zymodeme in a dog in Morocco. The isolated strain was first identified by the use of genotyping markers and confirmed by isoenzyme analysis. Phylogenetic analysis with the use of concatenated sequences from 26 Leishmania donovani complex strains revealed strong geographical correlation with the MON-24 strain from Morocco clustering with other East African strains whereas two other MON-24 strains clustered with L. infantum strains. Interestingly, the two distinct populations of MON-24 identified with the use of genotyping markers cannot be distinguished by multilocus enzyme electrophoresis.

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).

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.

Leishmaniosis due to Leishmania infantum in a FIV and FelV positive cat with a squamous cell carcinoma diagnosed with histological, serological and isoenzymatic methods.

Leishmaniosis caused by Leishmania infantum is an endemic zoonosis present in the Mediterranean area. Canidae (dog and fox) constitute the main reservoir hosts for the parasite, whilst wild rodents or the cat can be carriers of the protozoan and are considered as secondary potential reservoirs. This paper describes a case of disseminated feline leishmaniosis with cutaneous (ulcerative), visceral (spleen and lymph nodes) and blood involvement in a FIV-FelV positive cat. The microscopic identification of the Leishmania infection was initially made on a skin biopsy of the temporal area, where a squamous cell carcinoma was diagnosed. The diagnosis of the disease was achieved by several serological techniques (ELISA, IFAT and Western-blot). The strain was obtained by blood culture, characterized by electrophoresis of isoenzymes and identified as Leishmania infantum zymodeme MON-1. Since the infection due to L. infantum is a zoonosis, the potential feline reservoir should be more investigated. Serological analysis by Western blot on domestic cats provides a useful tool. In veterinary practice, feline leishmaniosis should be systematically included in the differential diagnosis when compatible cutaneous lesions are present, especially in the endemic areas of canine leishmaniosis.

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

The histological spectrum of visceral leishmaniasis caused by Leishmania infantum MON-1 in acquired immune deficiency syndrome.

Visceral leishmaniasis (VL) due to Leishmania infantum is endemic in Southern France and can be considered as an opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). Co-infection with Leishmania sp. and human immunodeficiency virus (HIV) is emerging, but pathological findings of leishmaniasis in AIDS have been poorly documented, and scattered case reports have include morphological descriptions. The clinicopathologic analysis of 16 patients with HIV and VL were evaluated. The clinical presentation was characteristic of VL, with fever, hepatosplenomegaly, and pancytopenia in 6 patients, and the diagnosis was confirmed by finding amastigotes of Leishmania sp. in bone marrow smears and biopsy specimens. In 4 patients, the initial diagnosis of VL was made fortuitously in gastrointestinal biopsies performed systematically (3 patients) or in case of diarrhea (1 patient). In one duodenal biopsy, Leishmania sp. and Mycobacteria sp. were associated. Liver biopsy allowed the diagnosis of VL in 3 cases. Autopsy was performed in 9 patients, showing a disseminated leishmaniasis with very unusual localizations (adrenal and heart) in 2 cases. Cutaneous leishmaniasis involvement was noted before (4 patients), at the same time (2 patient), or after (1 patient) the diagnosis of VL. Inflammatory infiltrates noted with Leishmania sp. infection were made by CD68 macrophages with (8 patients) or without (8 patients) associated CD8 positive lymphocytes. Immunoperoxidase study using polyclonal anti-Leishmania sp. antibodies contributed to the diagnosis in all cases. Electron microscopy of 2 digestive biopsy specimens showed the ultrastructural characteristics of Leishmania sp. amastigotes. The zymodeme MON-1 of L infantum was identified by isoenzyme electrophoresis in all patients. The mean of CD4 counts was 37/mm3 at the time of diagnosis, and the mean duration before the death was 8 months. As shown in this study, VL in AIDS can be diagnosed in gastrointestinal or liver biopsies. Diagnosis of VL was made when the CD4 count was very low and was correlated with a poor prognosis.

Short report: Treatment failure due to mixed infection by different strains of the parasite Leishmania infantum.

Therapeutic failure in a human immunodeficiency virus-negative patient with visceral leishmaniasis was due to mixed infection by two different Leishmania infantum zymodemes: L. infantum zymodeme MON-98, which is a rare zymodeme and is reported for the first time in Greece, and zymodeme MON-1, which is common in the Mediterranean region. The two strains were isolated from two samples of bone marrow from the patient obtained before the administration of treatment and 20 days later, since there was no improvement in the clinical signs. The zymodemes MON-98 and MON-1 exhibited different behaviors in vitro and showed different sensitivities to meglumine antimoniate in vitro and in vivo, as shown by clinical findings. Mixed infections with different Leishmania strains may explain the differences in the clinical course of leishmaniasis in many patients and may be the reason for treatment failures.

Experimental pathogenicity of viscerotropic and dermotropic isolates of Leishmania infantum from immunocompromised and immunocompetent patients in a murine model.

The pathogenicity of 22 strains of Leishmania infantum from 11 HIV-infected and 11 immunocompetent patients with visceral (VL, n = 16) or cutaneous (CL, n = 6) leishmaniasis, belonging to 3 zymodemes (MON-1, n = 14; MON-29, n = 5; MON-33, n = 3), was studied using a murine model. For each strain 16-20 BALB/c mice were infected at day 0 (d0) by i.v. injection of 10(7) stationary-phase promastigotes. Parasite burdens were quantified in the spleen and liver of 4-5 mice of each strain at d7, d20, d60 and d90 or d100, using a sensitive culture microtitration technique. A great variability of infection profiles between strains was observed: (i) six strains showed a progressive infection, with a predominance of hepatic parasites at d7 or d20 (10(4)-10(6) g-1), then a continuous rise of splenic parasites reaching 10(5)-10(7) g-1 at d90 or d100 contrasting with a stagnation or decrease in the liver; (ii) ten strains gave a controlled infection with hepatic parasite burden reaching 10(4)-10(5) g-1 at d7 or d20, followed by a more or less rapid decline leading frequently to no detectable parasites; (iii) six strains resulted in other profiles, i.e., undetectable infection (n = 1) or low parasite loads (n = 4), or late occurrence of parasites in the spleen (n = 1). No relationship was observed between profile and growth characteristics in vitro or zymodeme of the strain. Strains originating from CL never gave a visceralizing pattern in mice, but belonged more frequently to the avirulent type compared to VL strains. Strains from HIV-infected patients were not less virulent than those from immunocompetent individuals. These results showed that the course of L. infantum infection varies markedly with intrinsic parasite factors that display striking intraspecific variability.

Cutaneous leishmaniasis due to Leishmania infantum. Case reports and literature review.

BACKGROUND: Leishmania infantum recently has been identified as a possible agent of cutaneous leishmaniasis (CL). This species has been isolated from cutaneous lesions of patients from the Mediterranean Basin. However, little is known about the clinical, biological, or therapeutic features of this newly recognized CL. OBSERVATIONS: Six patients aged 9 months to 85 years in southeastern France were found to have autochthonous leishmaniasis. Parasitological identification showed that the agent was L infantum, zymodemes Montpellier-1 for 2 patients and Montpellier-24 for 1 patient. Five patients who underwent testing with a Western blot assay were found to have antibodies against 4 antigens with molecular masses of 18, 21, 23, and 31 kd. Five patients were successfully treated with local injections of N-methylglucamine, and 1 patient was successfully treated with topical paromomycin sulfate. No patient had visceral disease at diagnosis or after follow-up. CONCLUSIONS: Recent data provide increasing evidence that L infantum is an important agent of CL. In southwestern Europe, this species is the only agent that has long been identified from autochthonous CL. Leishmania infantum should be considered an agent of CL in areas in which visceral leishmaniasis is endemic. Western blot assay could be a useful test for the diagnosis, but precise parasitological identification is important to having a better knowledge of the disease. The relationships between CL and the visceral disease have to be explored.

Phlebotomus (Phlebotomus) duboscqi (Diptera: Phlebotominae), naturally infected with Leishmania major in southern Ethiopia.

In a survey of Leishmania infections in phlebotomine sandflies in visceral leishmaniasis focus at Aba Roba, Segen Valley, southern Ethiopia, Phlebotomus duboscqi was found naturally infected with L. major (zymodeme MON-74). This is the first isolation of L. major from a vector sandfly in Ethiopia, from an area where human and/or rodent infections with L. major are, so far, unknown.

Leishmaniasis due to Leishmania tropica MON-102 in a new Moroccan focus.

In Morocco, between March and April 2002, large numbers of human leihsmaniasis cases were detected during a survey at Zouagha My Yacoub province in Fès State. Among 95 cases, 76 were positive by direct observation of Giemsa-stained smears. Sixteen stocks were isolated in NNN medium and identified as Leishmania tropica MON-102, using isoenzyme techniques on starch gel electrophoresis.

Epidemic visceral leishmaniasis in southern Sudan: identity and systematic position of the parasites from patients and vectors.

Twenty-five strains of Leishmania donovani isolated from humans and Phlebotomus orientalis in an epidemic area of southern Sudan were shown to belong to 3 very similar zymodemes: MON 18 (11 strains), MON 30 (1 strain) and MON 82 (13 strains). The 3 zymodemes are very closely related and seem to behave as a single population.

Delayed culture of Leishmania in skin biopsies.

Between January 1997 and October 1998, 16 skin biopsies collected from 13 patients with cutaneous leishmaniasis in French Guiana were inoculated in culture medium after travel for 3-17 days from the place of biopsy to the culture laboratory in France. Each biopsy fragment was introduced near the flame of a Bunsen burner into the transport medium (RPMI medium supplemented with 10% fetal calf serum) which was maintained at ambient temperature during postal delivery to France. In France the biopsies were ground in sterile saline before being inoculated into NNN culture tubes. The cultures were incubated at 25 degrees C and subcultured every week until the 5th week. The cultures were positive in 9 cases, remained negative in 4, and were contaminated in 3 cases. Positive results were obtained at all seasons and for 3 different Leishmania species. The study indicates that delayed culture can yield useful results from biopsies taken in field conditions.

Leishmania species, drug unresponsiveness and visceral leishmaniasis in Bihar, India.

Sixteen isolates obtained, in January 1998-December 1999, from splenic aspirates from sodium stibogluconate-resistant cases of visceral leishmaniasis (VL; Indian kala-azar) and drawn from different districts of Bihar (India) were identified as Leishmania donovani. By isoenzyme analysis, all the strains were found identical to the WHO reference strain L. donovani MON-2 and differed from L. tropica MON-5. This study suggested that resistant cases of VL in Bihar were caused by L. donovani and not by L. tropica. No new strain responsible for drug unresponsiveness emerged during this period and other cause or causes of emergence of drug resistance should be sought. All the patients were cured with amphotericin B.

Human visceral leishmaniasis in Alpes-Maritimes, France: epidemiological characteristics for the period 1985-1992.

In 8 years (1985-1992), 65 cases of human visceral leishmaniasis (HVL) have been diagnosed in the department of Alpes-Maritimes, France, 56 of them having been infected locally. The annual frequency has increased from 3 cases in 1985 to 15 cases in 1992. There is a low rate of paediatric cases (29%) and a predominance of males among adult cases (85%). Since 1986, 19 cases of co-infection with Leishmania and human immunodeficiency virus 1 have been reported, corresponding to 40% of adult cases and to 30% of the total cases. The frequency of co-infections is stable at about 3 per annum. Isoenzymatic identification of the strains isolated from patients confirmed Leishmania infantum zymodeme MON-1 as responsible for most if not all HVL in the department of Alpes-Maritimes; 42 of the 44 strains isolated belonged to that zymodeme.

The use of isoelectric focusing on polyacrylamide gel for the enzymatic analysis of 'Old World' Leishmania species.

Using isoelectric focusing (IEF), a high resolution electrophoresis technique, we analysed 6 enzymes of 24 cloned strains representing all major taxa of 'Old World' Leishmania. The comparison of enzymatic patterns obtained with IEF and starch gel electrophoresis showed that IEF is a more discriminatory and more informative technique for the enzymatic analysis of Leishmania strains; it can detect very slight differences between 2 electromorphs not revealed with starch gel electrophoresis. Moreover, IEF detected several multi-banded patterns which appeared as single bands with starch gel electrophoresis. These multi-banded patterns could not be the result of strain heterogeneity since all the strains had been cloned. Their significance in the biology of Leishmania is discussed.