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.     

Human cutaneous leishmaniasis due to Leishmania infantum in the Sidi Bourouis focus (Northern Tunisia): epidemiological study and isoenzymatic characterization of the parasites

The authors report an increase of the number of case of cutaneous leishmaniasis in the Sidi Bourouis region community of Siliana Governorate (Tunisia), with 38 cases diagnosed in 6 months (1st November 2000-30th April 2001), contrary to its usual sporadic character. The isoenzymatic identification of 15 isolated strains emphasizes the role of the Leishmania infantum zymodeme MON-1 as an important factor in the genesis of the sporadic cutaneous leishmaniasis of Northern Tunisia. In fact it was isolated in 6 cases while L. infantum MON-24, the usual agent was isolated in 9 cases.     

Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST)

Leishmaniasis caused by Leishmania infantum is a prevalent disease in dogs and humans. A serological survey of Leishmania infection in dogs was carried out in the endemic region of Alto Douro (north Portugal). Two hundred and ninety-four dogs from the municipality of Peso da Régua were examined for clinical signs of canine leishmaniasis (CanL), and sera samples were evaluated by the direct agglutination test (DAT) and the fast agglutination screening test (FAST). The sero-prevalence of infection was 20.4%, after screening the study population by FAST and subsequent confirmation by DAT. The overall prevalence of disease was 3.1%. Only 15.0% of the sero-positive dogs had clinical signs of CanL. A high degree of agreement (88.4%; kappa value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test for canine Leishmania infection in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of CanL.     

Re-emergence of visceral and cutaneous leishmaniasis in the Greek Island of Crete

Leishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50?m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica).     

Cutaneous leishmaniasis in Tunisia: results of the iso-enzymatic characterization of 71 strains

Three clinico-epidemiological forms of cutaneous leishmaniasis (CL) exist in Tunisia: zoonotic cutaneous leishmaniasis (ZCL; epidemic in the centre and the south-west); sporadic cutaneous leishmaniasis (SCL; found in the north); and chronic cutaneous leishmaniasis (CCL; originally described from Tataouine, in the south-east). As few isolates of Leishmania from Tunisian cases of CL have been typed, isolates were collected, using NNN medium, from 71 such cases. Most (59) of the cases investigated came from the north of the country, including 16 from Sidi Bourouis, where there was an epidemic outbreak of SCL in early 2001; the other 12 cases were natives of the centre or south of the country. The 71 strains were then characterized, at the Centre National de Référence des Leishmania, in Montpellier, France, by iso-enzyme analysis. This revealed four zymodemes: two of L. infantum and one each of L. major and L. killicki. The MON-1 zymodeme of L. infantum, which is more usually associated with visceral leishmaniasis, was recovered from seven of the cases, including six natives of Sidi Bourouis. The MON-24 zymodeme of this species, which appears to be responsible for the SCL, was isolated from 48 cases, all of whom lived in the north of the country. Another 15 cases (nine from the centre, four from the north, and two from the south-east of the country) were found to be harbouring L. major MON-25, the zymodeme usually causing ZCL. Only a single isolate of L. killicki was made; this was of the MON-8 zymodeme responsible for the CCL, and came from a native of Gafsa, in the south-west. Six of the cases investigated (five infected with L. infantum MON-24 and one with L. major MON-25) showed involvement of their nasal and labial mucosae. These results increase the number of strains typed from Tunisian cases of CL more than four-fold, and should help to elucidate the geographical distribution and epidemiology of the various forms of the disease.     

The identification and variability of the parasites causing leishmaniasis in HIV-positive patients in Italy

Between 1988 and 1998, 258 Leishmania strains from patients infected with HIV were characterized by iso-enzyme electrophoresis at the Istituto Superiore di Sanità (ISS) in Rome. Most (227) of the isolates came from 80 Italian patients with visceral leishmaniasis (VL), the rest from cases of Leishmania/HIV co-infection in other Mediterranean countries. Every strain was found to be Leishmania infantum. In Italy, 19 zymodemes of L. infantum were identified, broadly divided into three groups. Over 50% of the Italian patients were infected by the commonest agent of Mediterranean VL in HIV-negative individuals (zymodeme MON-1) whereas the remaining patients were infected by two distinct groups of zymodemes: one usually causing simple, self-resolving cutaneous leishmaniasis in HIV-negative patients, the other consisting of agents detected, so far, only in HIV-positive subjects. This last group, consisting of seven zymodemes, showed re-assortment patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. A similar grouping was also observed in the isolates from other Mediterranean countries. Basing on general data recorded at the ISS over the last 20 years, accurate identification of the geographical origin of the zymodemes was attempted by careful analysis of the patients' histories, using the iso-enzyme electromorphs as geographical markers. Furthermore, a polymorphism index (no. of zymodemes/no. of patients) was defined for each Leishmania species and geographical region, and used to assess the level of L. infantum zymodeme heterogeneity in Italy, before and after stratification by HIV status. The greatest zymodeme heterogeneity was found in Sicily (southern Italy), with nine zymodemes identified among only 30 HIV-positive patients.     

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.     

Palestinian infantile visceral leishmaniasis caused by a genetic variant of Leishmania infantum belonging to a new zymodeme

The parasites causing a Palestinian case of infantile visceral leishmaniasis (IVL) and those from four dogs from the Jenin District were identified serologically, biochemically and molecular biologically as Leishmania infantum, showing dogs act as a reservoir. The strain from the human case was distinct because of its unique 200-bp kDNA-polymerase chain reaction (PCR) component in its restriction fragment length polymorphism (RFLP) profile after digestion with the endonuclease RsaI, and by the electrophoretic mobility of its malate dehydrogenase (MDH(140)), designating it the reference strain of a new zymodeme of L. infantum, MON-281.     

Canine Leishmania infantum enzymatic polymorphism: a review including 1023 strains of the Mediterranean area, with special reference to Algeria

This bibliographic review reports the isoenzyme polymorphism of 1023 Leishmania infantum strains isolated from dogs that have been characterized by multilocus enzyme electrophoresis in the Leishmania Reference Centre of Montpellier, or in other laboratories, to which this typification technique has already been transferred. Between 1981 and 2010, a total of 12 zymodemes were identified around the Mediterranean basin: MON-1, MON-24, MON-34, MON-72, MON-77, MON-80, MON-98, MON-105, MON-108, MON-199, MON-199 var NP1130 and MON-281, of which 6 were present in Algeria. The zymodeme MON-1 was predominant (86.5% of the strains). The dog was confirmed as the main reservoir of L. infantum MON-1, while the reservoir of the other zymodemes has not yet been identified. The enzymatic polymorphism is relatively high in Algeria and in Spain in contrast to other Mediterranean countries. The reasons for this polymorphism are discussed.     

Relapses versus reinfections in patients coinfected with Leishmania infantum and human immunodeficiency virus type 1

In the Mediterranean basin, Leishmania infantum is a major opportunistic parasite in people with acquired immunodeficiency syndrome (AIDS), and up to 9% of the patients with AIDS suffer from newly acquired or reactivated visceral leishmaniasis. Distinguishing between reinfections and relapses in these patients is important because some apparent treatment failures occur in patients with new rather than reactivated infections. Isoenzyme characterization is limited for use in determining relapsed versus newly acquired leishmaniasis in human immunodeficiency virus (HIV)-infected patients because of the variability of L. infantum and the predominance of the MON-1 zymodeme in people coinfected with HIV. A seminested polymerase chain reaction (PCR) was used to amplify L. infantum minicircle kinetoplast DNA, and, after digestion, the restriction fragment-length polymorphism (RFLP) profiles showed that 3 (7.5%) of 40 patients coinfected with L. infantum and HIV had a new infection, whereas isoenzyme characterization indicated that all 40 patients had infection relapses. These results suggest the utility of this PCR-RFLP analysis in detecting leishmaniasis reinfection in HIV-positive patients.     

Will the introduction of Leishmania tropica MON-58, in the island of Crete,lead to the settlement and spread of this rare zymodeme?

The rare zymodeme, Leishmania tropica MON-58, was isolated from a young Afghan refugee with a facial cutaneous lesion who had come to live in Crete early 2008. The same zymodeme variant was isolated from a local dog that had never travelled outside the island, with symptoms of visceral leishmaniasis, which stayed in the area where the patient worked during the summer months. This is the first record of L. tropica in a host, other than human, in Greece and another example of introduction of a vector borne pathogen in a focus where local vector/s can sustain it, with the risk of initiation of new transmission cycle/s.     

Transmission of Leishmania infantum MON-98 to hamsters by the bite of Phlebotomus langeroni Nitzulescu (Diptera: Psychodidae)

The ability of Phlebotomus langeroni to successfully acquire and transmit Leishmania infantum MON-98 to hamsters was demonstrated. Sand flies and Leishmania both originated from an infantile visceral leishmaniasis focus in El Agamy Egypt. P. langeroni females were infected by feeding on lesions of needle-inoculated hamster and on infected blood suspension using a chick-skin membrane apparatus. Infection rate of sand flies fed on membrane was 88% compared to 7.8% for flies fed on leishmanial lesion. The transmission to hamster took place by the bites of infective flies taking a second blood meal, on the 8th to 10th day post-feeding. Furthermore, successful transmission was by the bites of flies that took no blood or that took full blood meal. Whereas flies that took full blood meal were not infective as indicated by dissection. In three hamsters, lesions developed after three months. Leishmania amastigotes were demonstrated from the lesion as well as from the liver and spleen of infected hamsters.     

Leishmaniases and the Cyprus Paradox

In Cyprus, leishmaniasis has been considered exclusively a veterinary problem. It was prevalent before 1945, and until its recent reemergence, it was nearly eradicated by 1996 as a consequence of the destruction of reservoir hosts and vectors. A survey carried out to provide an unbiased estimate of current transmission rates in dogs and humans showed a 9-fold increase in dog seroprevalence (reaching 14.9%) compared with 10 years ago. However, no human cases caused by Leishmania infantum were detected, although L. donovani cases were reported recently. The 62 strains isolated from dogs were typed as L. infantum MON-1 (98.4%), which is the predominating zymodeme in the Mediterranean region, and MON-98 (1.6%). The Phlebotomus species P. tobbi (vector of L. infantum in Cyprus), P. galilaeus, and P. papatasi were the predominant species captured. Two transmission cycles seem to run in parallel in Cyprus: in dogs with L. infantum and in humans with L. donovani.     

Localized mucosal leishmaniasis due to Leishmania (Leishmania) infantum: clinical and microbiologic findings in 31 patients

The clinical and microbiologic characteristics of 31 patients with mucosal leishmaniasis due to Leishmania (Leishmania) infantum are described. Twenty-eight (90%) patients were male. Mean age at presentation was 48 +/- 14 years. Thirteen (42%) patients had no underlying disease, while 18 (58%) patients had several other medical conditions. Fifteen (48%) patients were immunocompromised, 7 patients were infected with human immunodeficiency virus (HIV), and 3 were graft recipients. The primary location of lesions was the larynx in 11 (35%) patients, oral mucosa in 10 (32%) patients, and the nose in 5 (16%) patients. Mucosal lesions were painless in all patients but 2 and consisted of whitish, red, or violaceous nodular swelling or tumorlike masses. Ulceration was reported in 6 patients. Pathologically, the lesions showed a chronic inflammatory infiltrate. Granuloma may be seen.The localization of the lesions determined the symptomatology of the disease. Symptoms included hoarseness, difficulty swallowing, and nasal obstruction. The disease presentation was usually protracted, with a mean time from the onset of symptoms to diagnosis of 13 months (range, 3 wk-4.5 yr), and the clinical diagnosis was usually mistaken for neoplasia of the upper aerodigestive tract. No laboratory abnormalities were found in these patients due to the localized disease, apart from those attributed to underlying diseases.Parasites were easily identified in smears or sections by Giemsa stain or hematoxylin-eosin stain. Leishmania was grown in culture in 12 (60%) patients; culture was negative in 8 (40%) patients. Leishmania (Leishmania) infantum was identified in only 9 instances. The following zymodemes were reported: MON-1 (2 patients), MON-24 (2 patients), MON-27 (1 patient), and MON-34 (1 patient). Serologic test results were known in 25 patients. Serology was usually positive at low titer; 6 (24%) patients had negative serologic test results.Twenty patients were treated with antimonial compounds for between 3 and 36 days. Three patients were given drugs other than antimonial drugs. Five patients were treated only locally, by surgery (3 patients) or topical medical therapy. One patient received no therapy, and treatment was not reported in 2 cases. Patients were cured in 25 (89%) cases, and sequelae were uncommon (14%). Relapse was detected in 2 individuals and 1 patient developed visceral leishmaniasis after treatment. Two HIV-coinfected patients died of causes unrelated to leishmaniasis.The results of the present report stress the clinical importance of searching for the presence of Leishmania in patients with suspected neoplasia of the upper respiratory tract if they have visited or resided in zones endemic for Leishmania (Leishmania) infantum. The treatment of choice for these patients is not established yet, but most patients respond to antimonial compounds given for 28 days or less.     

Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains

A series of 1048 Leishmania strains from Old World cutaneous leishmaniasis foci, isolated between 1981 and 2005, were studied by isoenzyme analysis. The strains were obtained from humans, rodents, dogs and sandflies from 33 countries. The four typically dermotropic species, Leishmania major , L. tropica , L. aethiopica and L. killicki , were found. The viscerotropic species L. donovani and L. infantum, which can occasionally be responsible for cutaneous leishmaniasis, are not considered in this paper. Leishmania major was the least polymorphic species (12 zymodemes, 638 strains). Leishmania tropica was characterized by a complex polymorphism varying according to focus (35 zymodemes, 329 strains). Leishmania aethiopica , a species restricted to East Africa, showed a high polymorphism, in spite of a limited number of strains (23 zymodemes, 40 strains). Leishmania killicki , mainly restricted to Tunisia had a single zymodeme for 39 strains. Recently a parasite close to L. killicki (one zymodeme, two strains) was isolated in Algeria, which lead us to revise the taxonomic status of this taxon.     

First report of natural infection of least weasel (Mustela nivalis Linnaeus, 1776) with Leishmania major in Tunisia

Zoonotic cutaneous leishmaniasis, caused by Leishmania major (L. major), is endemic in Tunisia. Several rodents have been identified as reservoir hosts of parasites. This study reports, for the first time, the natural infection with L. major zymodeme MON-25 in a specimen of least weasel: Mustela nivalis Linnaeus, 1776 (M. nivalis) collected in Sidi Bouzid. This finding justifies further research on larger samples of this animal to verify its role as a potential reservoir host for cutaneous leishmaniasis in Tunisia.     

Leishmaniasis in Sardinia. IV. Epidemiological appraisal of cutaneous leishmaniasis and biochemical characterization of isolates

Cutaneous leishmaniasis (CL) is endemic in Sardinia where 250 cases were reported from 1922 to 1988. The province of Sassari, in the north of the island, shows the highest number of cases. The vertical distribution of localities where cases had occurred is analysed and the age distribution of cases is given. Isolation of parasites was attempted in 12 cases, but successful growth of Leishmania was obtained from three cultures only. The stocks belonged to two different zymodemes of L. infantum: zymodeme Montpellier (ZMON) 24 and ZMON 24 MPI variant. The distribution of dermotropic L. infantum zymodemes in the Mediterranean area is discussed and the presence of a possible parasite reservoir is suggested.     

An outbreak of cutaneous leishmaniasis caused by Leishmania infantum in Geokcha? District, Azerbaijan SSR

A more than 60-case outbreak of cutaneous leishmaniasis was documented in the Geokcha? District of Azerbaijan between 1987 and 1988. The disease was noted in the rural areas only. All age groups were involved. The clinical picture of the disease was significantly different from anthropogenic cutaneous leishmaniasis caused by L. tropica but similar to cutaneous patterns caused by L. infantum which was a prevalent pattern in the southern France. Isoenzyme assay of the strain isolated in the Geokcha? causative agent defined it as Leishmania donovani sensu lato. Though an undoubted similarity of its enzymatic profile to L. infantum was observed it was distinguished as a singular zymodeme. A total of 9 Phlebotominae species were isolated in the Geokcha? District. Among them 5 species (P. kandelakii, P. tobbi, P. transcaucasicus, P. brevis, P. alexandri) were reviewed in the literature with regard to L. infantum transmission. Thus cutaneous leishmaniasis caused by L. infantum species in the rural area of the USSR was identified for the first time.     

Atypical lesions as a sign of cutaneous dissemination of visceral leishmaniasis in a human immunodeficiency virus-positive patient simultaneously infected by two viscerotropic Leishmania species

Leishmaniasis is considered an emerging opportunistic disease in human immunodeficiency virus (HIV)-infected patients who have considerably variable clinical presentation. We report a patient with visceral leishmaniasis who had unexpected clinical aspects (atypical cutaneous lesions appearing after long-term evidence of visceral parasites). The patient had hepatoesplenomegaly in the absence of fever, but was otherwise generally healthy. The HIV viral load was low despite severe immunossupression (low lymphocyte proliferation and low level of interferon-γ, concomitant with a high lymphocyte activation status). Surprisingly, two Leishmania strains were isolated from his bone marrow (typical L. infantum sequence MON-1, type A) and skin (L. donovani MON-2 sequence); this second strain had not been previously identified in Brazil. The association of visceral leishmaniasis and HIV/acquired immunodeficiency syndrome is a largely unknown disease, particularly in areas in which leishmaniasis is not endemic. Such atypical cases indicate that this disease can be undiagnosed in clinical settings.     

Liver involvement during visceral leishmaniasis

Visceral leishmaniasis is a life-threatening infection due to Leishmania parasites that has been reported in 62 countries. The Mediterranean area is endemic for Leishmania infantum and cases have been reported in Spain, France, Italy and Portugal. During the infection, parasites disseminate in the phagocyte cells of bone marrow, the spleen, liver and lymph nodes. In this paper, we review the clinical and biological signs observed in visceral cases of leishmaniasis with hepatic involvement. We also focus on experimental and pathophysiological data to clarify our understanding of liver involvement during this infection.