Surveillance of leishmaniases in France, 1999 to 2012.

Leishmaniasis is endemic in the south of France, where autochthonous disease is caused by Leishmania infantum, and affects both humans and dogs. The prevalence of canine leishmaniasis is between 3 and 66% depending on the region and the methods used. Human leishmaniases are also imported into France, mainly from French Guiana and North Africa. The surveillance of autochthonous and imported human leishmaniases is based on passive notification to the National Reference Centre for Leishmaniases (NRCL) created in 1998. Between 1999 and 2012, 317 autochthonous and 1,154 imported cases were notified to the NRCL. The average number of autochthonous cases notified per year was 22.6, mainly cases of visceral leishmaniasis (84.5%). All cases were infected in the south of France. Leishmaniasis incidence is 0.22 per 100,000 inhabitants in the endemic area. Imported cases were more frequent (annual mean of 82.4 cases) and consisted predominantly in cutaneous leishmaniasis (CL) cases (91%), essentially L. major CL imported from Maghreb and Sub-Saharan Africa, and L. guyanensis CL from French Guiana. This national notification system allowed a better understanding of the incidence and distribution of the disease; it is also useful to assess the temporal-spatial evolution of the disease in France, which appears relatively stable.

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.

[Conjonctival human myiasis by Oestrus ovis in Northern Africa]. / À propos de la myiase conjonctivale humaine à Oestrus ovis en Afrique du Nord.

Following the publication of a paper on Conjonctival human myiasis by Oestrus ovis in southern Tunisia by Anane and Ben Hssine (Bull Soc Pathol Exot (2010) 103(5):299-304), the author reminds that the discovery of this disease was made in Algeria, in 1907 by Edmond and Etienne Sergent.

[Georges Moustardier, physician of the Colonial Health Service and Overseas Pasteur Institutes and university professor]. / Georges Moustardier, médecin colonial, pastorien d'outre-mer et professeur d'université.

The purpose of this article is to provide a step-by-step description of Georges Moustardier's career. After completing studies at the Ecole Principale du Service de Santé de la Marine et des Colonies in Bordeaux, and at the Ecole d'Application du Service de Santé des Troupes Coloniales in Marseille, he was deployed to Indochina where he served as physician first at the Poulo Condor penitentiary from (1929 to 1930) and then in Cambodia from (1931 to 32). In 1933, he returned to Paris where he followed lectures on Microbiology at the Institut Pasteur, in Paris. He was then assigned to the Institut Pasteur in Madagascar from 1931 to 1932. From 1939 to 1944, he was Head of the General Hospital in Brazzaville, Congo and Director of the Medical School in French Equatorial Africa. He retired from the army in 1946. From 1949 to 1972, he held an academic position as Professor of Bacteriology at the Bordeaux School of Medicine.

Destruction of Leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers.

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 muM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

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.

The Sergent brothers and the antimalarial campaigns in Algeria (1902-1948).

Edmond and Etienne Sergent, "the Sergent brothers", were both born in Algeria. They both studied medicine at the Algiers Medical School and then followed the Course of Microbiology of Emile Roux at the Institut Pasteur in Paris (1899-1900). From 1900, they were put in charge of a permanent mission aimed at antimalarial control in Algeria, which was supervised by the Institut Pasteur. The first campaign was carried out during the summer of 1902 at a station of the East Algerian Railway Company. The success of this mission lead to the creation of the Antimalaric Department of Algeria in 1904, which was directed by Etienne Sergent for the duration his life. This antimalarial programme was progressively extended to many other locations. The programme was optimized between 1927 and 1947, in the experimental field study of the Ouled Mendil Marsh, where global environmental measures and drainage lead to settlement of farms, the families of which did not suffered from malaria. At a time when neither insecticides nor synthetic antimalarial drug existed, antimalarial control measures that were developed tended to target human reservoirs and the mosquito vectors. The extension of the programme across the Algerian territory lead to a decrease of both malaria endemicity and extension of affected areas.

[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).

[Edmond Sergent's discoveries on the vectorial transmission of agents of human and animal infectious diseases]. / Les découvertes d'Edmond SERGENT sur la transmission vectorielle des agents de certaines maladies infectieuses humaines et animales.

Edmond SERGENT has been head of the Institut Pasteur in Algeria from 1910 to 1963. During these years, he carried out an impressive scientific production and studied a lot of agents responsible for human, animal and plant diseases. In the field of vectorial transmission of infectious diseases, he made two essential discoveries: the transmission of cosmopolitan relapsing fever by human body louse in 1908, a year before Charles NICOLLE discovered the transmission of the classical exanthematic typhus by the same insect, and the transmission of cutaneous leishmaniasis by the phlebotomine sandfly. Moreover he made other discoveries in similar fields, such as the transmission of dromedary trypanosomiasis by Tabanids and later by stomoxys calcitrans, or the transmission of the pigeon Haemoproteus by Lynchia maura. Finally he described the transmission of Theileria dispar (now T. annulata) by the tick Hyalomma mauritanicum (1928).

[Cutaneous leishmaniases in French Guiana]. / Les leishmanioses cutanées en Guyane française.

Cutaneous leishmaniases are endemic over the entire territory of French Guiana. At least 5 distinct Leishmania species coexist in the sylvatic ecotopes of this French territory. The present paper checks the advances in the ecological research field during the past 5 years. The current epidemiological situation and trends are detailed successively Links between the recrudescence of leishmaniases and gold-mining are highlighted. The potential adaptation of the pathogenic complexes to the newly anthropized habitats is also described.

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.

Stages in the identification of phlebotomine sandflies as vectors of leishmaniases and other tropical diseases.

The first time the term phlebotomine sandfly was used, was by an Italian naturalist, Philippo Bonanni, in 1691. The first description though was made by another Italian naturalist, Scopoli, under the name Bibio papatasi. The name of the genus, Phlebotomus, was not given until 1840 by Rondani and Berté. The first description of an American phlebotomine sandfly was made by Coquillett, in 1907. The discovery of the three first sandflies in Brazil is the work of Lutz and Neiva, in 1912. From this date till 1921, 11 new species were described in this country and since then their number is still increasing and has reached 229 at this time. The history of the identification of phlebotomine sandflies as vectors, in Brazil like elsewhere in South America, is as complex as the one of the leishmaniases themselves, to which it is closely linked. The knowledge of cutaneous leishmaniasis in Brazil goes back to 1909, when Gaspar Vianna proposed to name the parasites that were found Leishmania braziliensis (1911). Following the observation of the Sergent brothers on the role of Phlebotomus papatasi in the transmission of cutaneous leishmaniasis in Algeria (1921), it became obvious that phlebotomine sandflies should be incriminated as vectors of cutaneous leishmaniasis in the Americas and that proof should be sought of their role. This is what various Brazilian scientists have done, like Aragão in 1922, Pessôa and Pestana in 1940. In the 1950s evidence was produced that the different forms of leishmaniasis that infest the American continent were caused by distinct species of the parasite. Subsequently, successful searches for the specific vectors were carried out. A by-product of the epidemiological studies of leishmaniases has been the discovery of the transmission of other parasites of the Trypanosomatid families (Crithidia, Endotrypanum, Trypanosoma). More recently, since the 1960s, a large number of viruses amongst which Rhabdoviridae, Bunyaviridae and Reoviridae, have been isolated from phlebotomine sandflies. Between 1961 and 1995, 69 serotypes of different arboviruses were obtained from different zones of Brazilian Amazonia.

Conserved linkage groups associated with large-scale chromosomal rearrangements between Old World and New World Leishmania genomes.

The genus Leishmania can be taxonomically separated into three main groups: the Old World subgenus L. (Leishmania), the New World subgenus L. (Leishmania) and the New World subgenus L. (Viannia). The haploid genome of Old World Leishmania species has been shown to contain 36 chromosomes defined as physical linkage groups; the latter were found entirely conserved across species. In the present study, we tried to verify whether this conservation of the genome structure extends to the New World species of Leishmania. 300 loci were explored by hybridization on optimized pulsed field gel electrophoresis separations of the chromosomes of polymorphic strains of the six main pathogenic Leishmania species of the New World. When comparing these New World karyotypes with their Old World counterparts, 32 out of 36 linkage groups were found conserved among all species. Four chromosomal rearrangements were found. All species belonging to the L. (Viannia) subgenus were characterized by the presence (i) of a short sequence exchange between chromosomes 26 and 35, and (ii) more importantly, of a fused version of chromosomes 20 and 34 which are separated in all Old World species. 69 additional markers were isolated from a plasmid library specifically constructed from the rearranged chromosomes 20+34 in an attempt to detect mechanisms other than a fusion or breakage: only two markers out of 40 did not belong to the linkage groups 20 and 34. On the other hand, all strains belonging to the New World subgenus L. (Leishmania) were characterized by two different chromosomal rearrangements of the same type (fusion/breakage) as above as compared with Old World species: chromosomes 8+29 and 20+36. Consequently, these two groups of species have 35 and 34 heterologous chromosomes, respectively. Overall, these results show that large-scale chromosomal rearrangements occurred during the evolution of the genus Leishmania, and that the three main groups of pathogenic species are characterized by different chromosome numbers. Nevertheless, translocations seem particularly rare, and the conservation of the major linkage groups should be an essential feature for the compared genetics between species of this parasite.

The 70-kDa heat-shock protein is a major antigenic determinant in human Trypanosoma cruzi/Leishmania braziliensis braziliensis mixed infection.

Five sera from Bolivian individuals chronically infected by Trypanosoma cruzi, and suffering an active Leishmania braziliensis braziliensis metastatic mucocutaneous lesion were characterized. They reacted with the T. cruzi recombinant antigens that are currently used as Chagas diagnostic reagents, and with several L. b. braziliensis proteins as assessed by Western blot. These sera showed an intense reaction with a T. cruzi and an L. b. braziliensis polypeptide of about 70 kDa. Expression cloning techniques demonstrated that the target of this immunologic reaction was a cross-reactive antigen, the 70-kDa heat-shock protein (HSP 70). High levels of anti-HSP 70 reactivity and positive reactions with all or some of the T. cruzi recombinant antigens JL7, JL8, and JL5, defined a serologic pattern that was characteristic of the T. cruzi/L. b. braziliensis mixed infection.

Cutaneous leishmaniasis in French Guiana: a review.

A review of a 10 year investigation carried out by various institutions on cutaneous leishmaniasis in French Guiana is presented, with emphasis on epidemiology, clinical aspects, and therapy.

Detection of circulating antibodies in cutaneous leishmaniasis by enzyme-linked immunosorbent assay (ELISA).

An immunoenzymatic diagnostic technique applicable to cutaneous leishmaniasis is described. The antigen used (Leishmania tropica major) was equally useful in diagnosing visceral and mucocutaneous forms of the disease. The criteria for positivity were defined by using groups of negative controls, and the specificity of the reaction was evaluated by using sera from patients with various diseases. Among these, sera from patients with lepromatous leprosy, tuberculosis, or African trypanosomiasis strongly cross-reacted with leishmania antigen. Examining serial dilutions of the sera facilitated the interpretation of the results and eliminated a significant percentage of false positives.

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.