Visceral leishmaniasis in hematopoietic cell transplantation: Case report and review of the literature.

Visceral leishmaniasis has been recognized as an opportunistic infection affecting people with cellular-immunity impairment, including hematopoietic cell transplantation (HCT) recipients. We describe the case of a young Italian man with Hodgkin lymphoma, who developed visceral leishmaniasis after multiple lines of chemotherapy and allogenic HCT. Literature review of visceral leishmaniasis in HCT recipients was also performed. Eleven patients (median age 50 years, 9 male) developed visceral leishmaniasis after allogenic (n = 9) and autologous (n = 2) HCT. Most of them presented with fever and pancytopenia. Bone marrow examination was the main diagnostic technique; liposomal amphotericin B was the treatment of choice. Four out of eight patients (for whom data are available) experienced visceral leishmaniasis relapse. Visceral leishmaniasis in HCT recipients is a rare event that should be suspected in patients with persistent fever, pancytopenia and possible exposure to Leishmania spp., remembering that - as well as South-East Asia, East Africa and South America - it is endemic in several European regions.

The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis.

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

Temporal trends in canine leishmaniosis in the Balearic Islands (Spain): a veterinary questionnaire. Prospective canine leishmaniosis survey and entomological studies conducted on the Island of Minorca, 20 years after first data were obtained.

Leishmaniosis is present in the Mediterranean region of Europe, where Leishmania infantum is responsible for the disease, dogs are the main reservoir, and sand flies of the Phlebotomus genus, subgenus Larroussius, are proven vectors. Some areas, including Minorca in the Balearic Islands, are considered free of the disease, despite the presence of vectors. However, in the context of the current expansion of canine leishmaniosis in parts of Europe, an epidemiological study using a veterinary questionnaire was carried out to establish the current situation of the disease in the Balearic Islands. While 50% of veterinarians thought that the incidence of canine leishmaniosis had not changed over time, 26.2% perceived an increasing trend, mainly those from Minorca, where most of the veterinarians polled (88.1%) considered the new diagnosed cases as autochthonous. A cross-sectional serological study performed in this island gave a seroprevalence rate of 24%. Seroprevalence among animals of local origin and with no history of movements to endemic areas was 31%. The presence of autochthonous canine leishmaniosis in Minorca was not correlated with an increase in vector density. The environmental and climatic factors that influenced the distribution and density of Phlebotomus perniciosus on the island and the possible causes of the apparent emergence of canine leishmaniosis in Minorca are discussed.

Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013.

An increased number of autochthonous visceral leishmaniasis (VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0-8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15-120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe.

The burden of visceral leishmaniasis in Italy from 1982 to 2012: a retrospective analysis of the multi-annual epidemic that occurred from 1989 to 2009.

Starting from 1989 Italy experienced an increase of visceral leishmaniasis (VL) cases over a baseline of 10 to 30 cases reported annually. The number of cases peaked in 2000 and 2004 with more than 200 cases/year, and subsequently declined to reach on average one third of the 2000 peak value in the period after 2010. A retrospective analysis from 1982 to 2012 showed that the multi-annual epidemic consisted of major components including (i) an outbreak involving infants and immunocompetent adults in parts of the Campania region (southern peninsular Italy) and that appears to have declined naturally, (ii) a second outbreak affecting human immunodeficiency virus (HIV)-infected individuals throughout the country, that declined owing to the use of highly active antiretroviral therapies (HAART), (iii) a generalised increase of VL cases in immunocompetent individuals and patients affected by associated conditions other than HIV from endemic regions of peninsular and insular Italy (other than Campania), which was due to a geographical spreading of VL foci, with no major case-clusters or outbreak features. A minor component consisted in the appearance of a few autochthonous cases in formerly non-endemic areas, starting from the early 1990s. Epidemic determinants and reasons for VL decline in the Campania region remain largely unexplained, despite the information available on canine reservoir and phlebotomine vectors in Italy.

The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region.

An updated view of the establishment and spread of the leishmaniases in Europe is presented, mostly with respect to newly emerging and re-emerging foci and the incrimination of neglected as well as new reservoir hosts. At the same time, a concept of specific versus permissive vectors reassesses the potential role of various sandfly species in Leishmania transmission and considers the risk of introduction of exotic Leishmania species in Europe. The leishmaniases are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural hosts and vectors, immunosuppressive conditions (like infection with human immunodeficiency virus (HIV) or organ transplantation-associated therapies in humans) and the consequences of war, all contribute to the transformation of the epidemiology of leishmaniasis. Such changes should be considered when studying the spread of the disease throughout Europe for targeted control measures to safeguard public health.

Recent advances in leishmaniosis in pet animals: epidemiology, diagnostics and anti-vectorial prophylaxis.

The leishmanioses are diseases caused by protozoa of the genus Leishmania, parasites infecting numerous mammal species, including humans, and transmitted by the bite of phlebotomine sand flies. They are a large group of diseases ranging over inter-tropical zones of America and Africa, and extend into temperate regions of Latin America, Europe and Asia. Pet animals are found infected with different Leishmania species but Leishmania infantum is the most widespread being dogs the main reservoir of zoonotic visceral leishmaniosis (ZVL). Dogs are very susceptible to this parasite and may suffer from a complex syndrome, canine leishmaniosis (CanL), one of the major zoonoses globally causing severe fatal disease in this animal. Infections in cats and horses have also been reported in areas where CanL is diagnosed. In Europe dogs and cats are common companion animals and their health is of great concern, therefore management of leishmaniosis in pets generally follows that of human ZVL. The recent spread of Leishmania infections in non-endemic territories has been monitored by means of canine surveys, which represent a suitable approach because of the dog's role as a sentinel host. New tools have been developed for the surveillance and control of ZVL. A number of insecticide-based preparations have been specifically registered for dog protection against sand fly bites, with elevated efficacy for both individual and mass protection.

Visceral leishmaniasis in Aba-Roba, south-western Ethiopia: prevalence and incidence of active and subclinical infections.

Between August 1997 and February 2005, a prospective study of human visceral leishmaniasis (VL) was undertaken in two villages in the Konso district of south-western Ethiopia, to provide epidemiological indices of subclinical infection and active VL. Six cross-sectional surveys at 6-month intervals (ending in August 2000) were complemented by a single survey in February 2005. The prevalences and incidences of leishmanial infection and active VL, which were determined using leishmanin skin tests (LST), direct agglutination tests (DAT) and parasitological diagnosis, varied spatio-temporally and by age and gender. At baseline, when 1339 individuals were investigated, the overall prevalences of LST positivity, DAT positivity and active VL among the 1232 subjects who had not been treated previously were 30.0%, 5.4% and 0.49%, respectively. During the study, <10% of the subjects found DAT-positive at baseline progressed to active VL (with a mean of about nine cases of subclinical infection for every one of active VL). The median age of an incident VL case was 10.5 years. The highest rates of LST conversion occurred among the subjects aged 5-25 years. A subject who became LST-positive during the study was much less likely to develop active VL than the other subjects.

Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan.

Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.

Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus (Diptera, Psychodidae) in a high-endemic focus of canine leishmaniasis in Rome province, Italy.

A 2-year survey aimed to study seasonal phenology, host-blood feeding preferences and Leishmania infections of Phlebotomus perniciosus, was carried out in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Sandfly densities were monitored by sticky traps, while CDC light traps and hand collections were used for individual sandfly analyses. Four species were identified, three belonging to Phlebotomus (P. perniciosus, Phlebotomus mascittii and Phlebotomus papatasi) and one to Sergentomyia (Sergentomyia minuta) genera. In sticky traps, P. perniciosus (53.5%) and S. minuta (46.0%) were the prevalent species, whereas specimens collected by CDC light traps and hand collections consisted mostly of P. perniciosus. The study showed a markedly different sandfly density between 2003 and 2002 collections (5024 specimens versus 644 specimens, respectively), which was attributed to different climatic conditions. In 2003 adults were active during 7 months (May-November) with a typical bimodal distribution, while in 2002 their activity was shorter (5 months, June-October) without clear density peaks. In this low-density year, the prevalence of P. perniciosus was significantly higher in domestic than in wild resting sites. Blood meal tests showed that P. perniciosus had no preferences for any of the two Leishmania susceptible hosts, being the forage ratio rate 0.7 and 1.0 for man and dog, respectively. A significant number of P. perniciosus was found with avian (60.3%) or ovine (24.2%) blood, that was in relation with the habitats where fed specimens were collected. The rate of specimens of P. perniciosus without blood and found positive for Leishmania DNA, detected by a sensitive nested-PCR technique, was much higher (27.6%) than the promastigote infection rate determined by microscopy in the dissected females (1.4%). The intense Leishmania transmission in the study area was confirmed by the high prevalence of anti-leishmanial antibodies in dogs (33.3%).

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

It presents a study about identification and variability of the parasites causing leishmaniasis in HIV-positive patients in Italy. Also brings too geographical origin of infection and geographical distribution of the enzymatic makers of Leishmania infantum. Document in PDF format, required Acrobat Reader.

Failure of a multi-subunit recombinant leishmanial vaccine (MML) to protect dogs from Leishmania infantum infection and to prevent disease progression in infected animals.

We report results of a Phase III trial of the multi-subunit recombinant Leishmania polyprotein MML for the protection of dogs against infection by Leishmania infantum. The antigen, also known as Leish-111f, is the first antileishmanial human vaccine entered Phase I clinical testing. The study was performed in a leishmaniasis endemic area of southern Italy. Three groups of 15 Leishmania-free beagle dogs each, received 3 monthly injections with vaccines A (MML+MPL-SE adjuvant), B (sterile saline = control) and C (MML+Adjuprime adjuvant), respectively, before transmission season 2002. The surviving dogs received a second three-dose vaccine course 1 year later. The dogs were naturally exposed to sandfly bites for 2.5 months in 2002, and for 5 months in 2003. Every 2 months post vaccination, dogs were examined by clinical and immunological evaluation, and by specific serology, microscopy, culture and PCR. A weak lymphoproliferative response to MML was seen in A and C groups throughout the study period. One year after the first vaccine course, the cumulative incidence of leishmanial infections was 40% in group A, 43% in group B and 36% in group C. Two-year post-vaccination (1 year after the second vaccine course) the cumulative incidence was 87% in group A (with three symptomatic cases), 100% in group B (with no symptomatic cases) and 100% in group C (with two symptomatic cases). The efficacy of the MML vaccine as an immunotherapeutic agent for the prevention of disease progression (subpatent infection-->asymptomatic patent infection-->symptomatic patent infection) was evaluated through follow-up of dogs found infected prior to the second vaccination. Among 15 infected animals, progression to a subsequent stage of infection was found in 5/6 dogs of group A, 3/6 of group B and 2/3 of group C. We conclude that vaccination with MML is not effective to prevent leishmaniasis infection and disease progression in dogs under field conditions.

Cutaneous leishmaniasis: an increasing threat for travellers.

Analysis of the literature on cutaneous leishmaniasis in low-prevalence countries suggests an increase in imported cases that is attributable to the growing phenomenon of international tourism, migration and military operations in highly endemic regions. Cases of imported cutaneous leishmaniasis are often missed initially, but diagnosis can be made non-invasively by PCR using skin scrapings of lesions as starting material. Cutaneous leishmaniasis is an emerging threat for travellers and should be considered in all patients presenting with slow-to-heal ulcers.

Entomological and parasitological study on phlebotomine sandflies in central and northern Albania.

An entomological survey was carried out in two districts of central (Kruje) and northern (Lezhe) Albania. Six collecting sites, showing a variety of diurnal resting sites, were monitored for adult sandflies from June through October 2002. Flies were collected with CDC miniature light traps, sticky traps and mechanical or hand aspirators in peridomestic sites, in bedrooms and inside cow barns, chicken coops and pigpens. All collecting sites monitored were found positive for sandflies. A total of 849 specimens were caught (29.2% males) belonging to five Phlebotomus species. Phlebotomus neglectus (75.6%) was the most abundant species followed by P. perfiliewi (14.4%), P. papatasi (4.6%), P. tobbi (3.6%) and P. similis (1.8%). The first adult of P. neglectus appeared on June 11 and the last one was collected on October 16. The highest density for this species was observed at the end of July. A total of 111 blood-fed females were caught from the two areas studied. P. neglectus was the only species found blood fed in Lezhe and the same species was prevalent (56.1%) in Kruje followed by P. perfiliewi (30.3%), P. tobbi (10.6%); P. papatasi was represented by only two specimens. Blood meal origin was determined in 45/66 (68.2%) of the females tested from Kruie district. P. neglectus was found fed on four hosts, showing the following feeding patterns: cow (71.4%), dog (117.1%), chicken (5.7%) and human (5.7%); P. perfiliewi was found fed on cow (80.0%) and chicken (20.0%), P. tobbi on cow (50.0%), chicken (25.0%) and dog (25.0%). One specimen of P. papatasi was found fed on cow. When such prevalences were analysed by the available biomass for each host present at the collecting site, P. neglectus resulted to be an opportunistic feeder rather than exhibiting preferences for any specific animal. PCR analysis of 39 P. neglectus from the Lezhe district gave negative results for the presence of Leishmania DNA.

[Monitoring of canine leishmaniasis in northern Italy: an update from a scientific network]. / Monitoraggio della leishmaniosi canina in nord Italia: aggiornamenti da un network scientifico.

Canine leishmaniasis (CanL) due to Leishmania infantum is a disease of great veterinary importance and a serious public health problem. In humans, L. infantum causes visceral (VL) and cutaneous leishmaniasis (CL) and the distribution of VL overlaps that of CanL. Currently, VL is considered by WHO as an emerging zoonosis in southern Europe. The dog is the only domestic reservoir of the infection and phlebotomine sandflies are the only proven vectors of leishmaniasis for dogs and humans. CanL is endemic in Italy, particularly in central and southern regions, including islands. Until 1983, all regions of northern Italy but Liguria and some territories of Emilia Romagna were considered free from CanL. From early '90s new stable foci of CanL have appeared, most of them located within classical endemic areas including territories of Emilia Romagna, Tuscany, Umbria, Marche, and Abruzzi regions. But the most relevant aspect, from an epidemiological point of view, has been the appearance of stable CanL foci in northern Italy, namely in Veneto and Piedmont regions. In these two foci, entomological surveys showed the presence of P. perniciosus and of a second phlebotomine vector, P. neglectus, which may have played a role in the CanL diffusion in some parts of northern Italy. Furthermore, in these areas, autochthonous human VL cases have occurred. There is therefore a realistic risk that CanL infection could rapidly spread through northern latitudes and a surveillance activity is strongly needed. For this reason, in October 2002, thanks to the collaboration and support of Intervet Italia, the network "LeishMap" was created, with the main purpose of monitoring the spread of CanL and vectors in northern Italy. LeishMap consists of scientific and sanitary institutions with proven experience both in field surveys and diagnostic methodologies on CanL and phlebotomine vector. It is organised in 4 Operational Units (OU), represented by researchers of the Veterinary Faculties of the University of Bologna, Padua, Milan and Turin, under the scientific coordination of the MIPI Department, ISS of Rome and with the collaboration of private and public veterinarians operating in the regions under study. During the first year of activity, each OU was involved in the serological and entomological surveillance of several territories in the respective regions, where recent autochthonous CanL cases were registered. The studies have involved five regions, namely Valle D'Aosta, Piedmont, Lombardia, Veneto, Trentino-Alto Adige and Emilia Romagna. In the Symposium 6 of this Congress we report detailed results of a retrospective analysis of data concerning CanL and vectors in northern Italy till 2002 and the preliminary results of 2003 on the seroprevalence rates observed in foci studied and on the entomological surveys carried out. In summary, the results outlined that already known foci of CanL are expanding from the original sites. Several new foci have been identified and many others are at high risk of evolving toward a stable endemicity. P. perniciosus has been found in all but one the suspected new foci. In Emilia Romagna region P. perfiliewi was identified in 2 areas and in one was the only species present. The occurrence of P. neglectus was confirmed in three regions, Veneto, Lombardia and Piedmont. In conclusion, from the 2002-2003 LeishMap activities it appears that further monitoring activities are necessary to identify new endemic foci of CanL, this representing the prerequisite for the implementation of programs for leishmaniasis control in northern Italy.

[Change in human visceral leishmaniasis treatment in Italy: retrospective study of 630 patients]. / Cambiamento nella terapia della leishmaniosi viscerale umana in Italia: studio retrospettivo su 630 pazienti.

Since the 1940s meglumine antimoniate (MA) has been the only first-line drug for visceral leishmaniasis (VL) treatment in Italy. From 1991 through 1994, several patients of all ages, representing 1/3 of all immunocompetent VL patients reported during that period, were enrolled in clinical trials of liposomal amphotericin B (L-AmB), which led to a novel, safe, short course of VL treatment as an alternative to MA. In the same period, other lipid-associated AmB drugs were registered in Italy for the treatment of fungal infections, i.e., AmB colloidal dispersion (ABCD) and AmB lipid complex (ABLC). A retrospective analysis was performed on data collected at the Unit of Protozoology of Istituto Superiore di Sanità, Rome, to assess whether changes have occurred in first-line drug regimens adopted in Italy for routine VL treatment, during the 1995-2002 period. The sample consisted of immunocompetent individuals clinically suspected for VL, in whom the disease was confirmed by the examination of serum and bone marrow specimens sent to the Unit by hospitals from throughout the country. Relevant information on patients was then recorded, which included drug regimens used and post-therapy results. We recorded treatment information for 630 patients, representing a large proportion (55.5%) of 1,135 immunocompetent individuals with VL reported in Italy from 1995 through 2002. About half were children (306). Every year, patients were referred by 19 to 42 hospitals, with a range of 1 to 30 patients per hospital. MA was the first-line drug used in 159 patients (25.2%). However, the proportion of MA-treated patients has steadily decreased from 55.9% in 1995 to 1.0% in 2002. We recorded the failure of MA therapy in 16 patients (10.1%), who were successfully retreated with a L-AmB regimen. The rate of MA failures significantly increased in recent years, from 5.3% in 1995 to 36.4% in 2000 (p = 0.01). AmB drugs have been the only alternative drugs used in the remaining 471 patients (74.8%). L-AmB accounted for most regimens (441, 93.6%). The proportion of patients treated with any AmB-based drugs increased from 44.1% in 1995 to 99.0% in 2002. Drug treatment was unsuccessful in 15 patients (3.2%), who were successfully retreated with a high-dose L-AmB regimen. This rate was significantly lower than the MA failure rate (p = 0.001). Results have shown a countrywide change in therapy over the period considered. A traditionally effective, but moderately toxic drug (MA) has been almost fully replaced by a new compound (L-AmB) with negligible toxicity, in an epidemiologic context of disease reemergence. Furthermore, short courses of 6 to 7 days, as required for lipid-associated AmB, are highly cost-effective if compared with 21- to 28-day courses needed for standard MA treatment.

[Parasite identification in the surveillance of imported leishmaniasis cases in Italy]. / Identificazione del parassita nella sorveglianza dei casi importati di leishmaniosi in Italia.

An accurate Leishmania classification was defined since 1980s by the use of isoenzyme analysis. To date, this procedure still represents the reference identification technique, despite the increasing use of molecular approaches. Studies and surveillance methods on leishmaniases are strongly conditioned by the knowledge and mapping of all the parameters characterizing each nosogeographical entity. On this respect, the identification of parasites from all the actors of the natural life cycle plays a key role. With the increasing population movements and climate changes, novel risk factors could be identified associated to Leishmania geographical distribution and spreading: a) the introduction into Italy of new populations of L. infantum from other countries; b) the introduction of new Leishmania species that may find a suitable milieau to support their life cycle in our country. The objective of this report is to present the surveillance activity on imported leishmaniases by the Leishmania Identification Reference Centre, ISS. Two different methodologies were routinely applied: a) isoenzyme electrophoretic analysis, which requires parasite culture, and b) a number of molecular techniques, used for both diagnosis and parasite identification, differently applied according to the geographical origin of the suspected leishmaniasis case. When possible, both types of methodologies were applied. From 1986 to June 2002, 38 imported cases of leishmaniases were identified: 9 visceral (VL) and 29 cutaneous (CL) cases, of which 22 from the Old and 7 from the New World. Pathology, Leishmania species/zymodeme and geographical origin features are reported in the paper. Seven out of 9 VL patients were HIV positive, of whom 5 detected in the period 1993-1995. This high importation rate can be associated to the general increase in Mediterranean Leishmania/HIV coinfections in that period. Following HAART treatment, VL imported cases became occasional; no introduction of new L. infantum populations has been detected. On the other hand, our findings show an increase of CL imported cases from different areas of the Old and New Worlds. This phenomenon, however, is so far limited to new Leishmania species that could hardly be introduced in our country, because of their strict biological requirements (i.e. vectors and/or natural reservoir hosts). Since June 2002, 10 further suspected imported cases were recorded. For these patients--whose Leishmania identification is still in course--the origin/visited geographical areas were only slight different from the previous. However, the characteristics of the patients are changing: there are more immigrants that occasionally visited their place of origin, and Italian military staff.

[Infantile visceral leishmaniasis in the Campania region, Italy: experience from a Paediatric Referral Centre]. / Leishmaniosi viscerale infantile in Campania: l'esperienza del Centro Pediatrico di Riferimento.

In the first half of the 20th century, visceral leishmaniasis (VL) was a common infantile syndrome in coastal territories of the Campania region of Italy. After World War II, the incidence dropped to a few cases/year for three decades; in late 1980s the disease reemerged among both children and adults. To face the VL recrudescence, a Paediatric Reference Centre was established at the Santobono-Pausilipon hospital in Naples, for the clinical diagnosis, care and drug treatment of all infantile VL cases occurred in the Campania region. Rapid laboratory diagnosis was secured by a Diagnostic Reference Centre established at the Istituto Superiore di Sanità. Here, we report on the epidemiological and parasitological features of all cases referred to the Centre in the past 15 years. From 1990 to March 2004, a total of 255 cases were diagnosed and treated at the Centre. The Figure shows the yearly trend of patients (min. 3 cases in 1990 and 1991, max. 30 cases in 2000). There were 135 males (52.9%); the age ranged 4 months-14 years, but 189 patients (74.1%) were < or = 3 years old. The majority of the patients (189, 74.1%) were from the Naples province, with a cluster of 102 cases (40% of total patients) from the towns and districts surrounding Vesuvius. Twenty-seven cases (10.6%) were from the town of Maddaloni, Caserta province, whereas 15 cases (5.9%) were from coastal villages of the Salerno province. Only 1 and 2 cases were from Benevento and Avellino provinces, respectively. All patients but seven, who have been treated with antimonial drugs in the 1990-1993 period, were successfully treated with a liposomal amphotericin B regimen. From bone-marrow aspirate samples, 138 Leishmania cultures were obtained in EMTM and Sloppy Evans' media, of which 134 have been typed by the electrophoretic analysis of 13 isoenzymes. Two zymodemes (Z) of L. infantum were routinely identified over the study period, ZMON-1 (the commonest zymodeme in the Mediterranean area) and ZMON-72, variant from MON-1 in PGM mobility and detected only in our region. The latter, identified in 61 patients (45.5%), was found exclusively distributed in towns of the Vesuvius area and in Maddaloni until 1996, but in recent years it appears to have spread to other areas of the Naples (including the island of Ischia) and Caserta provinces, but not to Salerno province. In conclusion, the VL macrofocus of the Naples-Caserta area is probably responsible for the highest number of infantile cases among any VL macrofoci described in southern Europe. Considering the limited efforts paid to control the canine reservoir, rapid diagnosis and appropriate treatment of patients still remain the first-line control measures aimed at reducing the health impact of the disease.

The isolation of Leishmania tropica and L. aethiopica from Phlebotomus (Paraphlebotomus) species (Diptera: Psychodidae) in the Awash Valley, northeastern Ethiopia.

In a survey of Leishmania infections in phlebotomine sandflies in a highly suspected focus of leishmaniasis in the Awash Valley (northeastern Ethiopia) between January 1994 and August 1997, a total of 3307 females of 11 Phlebotomus species (P. orientalis, P. fantalensis, P. saevus, P. sergenti, P. gemetchi, P. alexandri, P. bergeroti, P. duboscqi, P. arabicus, P. martini, and P. rodhaini) were dissected. Promastigotes were detected in 17 females of three species (11 P. saevus, 4 P. sergenti and 2 P. arabicus). Of these, only two P. saevus (one from Upper Awash and one from Middle Awash) and three P. sergenti (from Upper Awash) positives were successfully isolated in culture and were typed by isoenzyme analysis. Four isolates (two each from P. saevus and P. sergenti) were identified as new zymodemes (Z) of L. tropica and one isolate from P. sergenti was typed as a new zymodeme of L. aethiopica. This is the first finding of natural infections of P. saevus and P. arabicus and the first evidence for the former to be a vector of L. tropica. This is also the first time P. sergenti has been implicated in L. tropica transmission in Ethiopia; the isolation of L. aethiopica from a Paraphlebotomus species (P. sergenti) is also a new record. The possible presence of human cutaneous leishmaniasis (L. tropica and L. aethiopica), and wild reservoir host(s) of the parasites, especially rock hyrax (Procavia capensis) in the Upper and Middle Awash Valley remain to be determined.

Cutaneous leishmaniasis in Multan: species identification.

OBJECTIVE: To identify the Leishmania species being responsible for cutaneous leishmaniasis in Multan. METHOD: Parasites were isolated from clinically and parasitologically confirmed lesions of cutaneous leishmaniasis from 30 patients by fine needle aspiration (FNA). The bioptical materials were then cultured in Evans Tobie's medium and parasites isolated were identified by isoenzyme electrophoresis technique. RESULTS: Successful Leishmania isolates were obtained from 16 patients. All strains were identified by biochemical techniques as belonging to Leishmania tropica zimodeme MON7 variant PGD. CONCLUSION: The causative species was identified as Leishmania tropica.