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.

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.

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.

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.

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%).

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.

Epidemiological surveillance of leishmaniasis in HIV-1-infected individuals in Italy.

OBJECTIVE: To actively detect leishmaniasis in HIV-1-infected individuals in Italy, to describe the epidemiological features of the disease in these patients, and to compare them with epidemiological features of leishmaniasis in HIV-negative patients. DESIGN: Retrospective and prospective surveillance study. PATIENTS: Italian patients with HIV-1 infection and leishmaniasis diagnosed between 1985 and 1994. RESULTS: We recorded 116 leishmaniasis cases (115 visceral leishmaniasis), of which 94 (81%) were diagnosed over the last 4 years. Seventy-eight patients (67%) fulfilled the 1993 Centers for Disease Control and Prevention AIDS criteria. Leishmaniasis was passively reported in only 18% of cases. Leishmania incidence estimated among approximately 2700 AIDS patients living in leishmaniasis endemic areas averaged 1.6%, with a maximum of 4.9% in Sicily. These rates were up to 500-fold higher than among HIV-negative individuals living in the same areas, and were similar to those of ubiquitous opportunistic agents indicative of AIDS condition. Data from two major endemic regions indicated that overlap of HIV-1 and Leishmania infections has focal characteristics. The occurrence of small case clusters would suggest occasional modes of Leishmania transmission different from the insect vector. The isoenzyme characterization of 38 Leishmania stocks showed a zymodeme spectrum qualitatively and quantitatively different from that of the parasitic agent of visceral leishmaniasis in HIV-negative adults. CONCLUSIONS: Active surveillance provided reliable evaluation on the occurrence of HIV-Leishmania coinfections in Italy, although it was limited to hospital-based cases in this study due to general under-reporting of cases. Biological and epidemiological spectrum of the disease suggests that visceral leishmaniasis should be included among AIDS-defining pathologies.

A kinetoplast DNA probe diagnostic for Leishmania major: sequence homologies between regions of Leishmania minicircles.

A restriction fragment from a cloned kinetoplast minicircle DNA has been shown to be diagnostic for Leishmania major. This 402-bp TaqI fragment has been used routinely (as a radiolabelled probe) to detect 10(4) parasites in simple dot blots, both experimentally and in epidemiological surveys. It positively identified all stocks of L. major tested (including all six known zymodemes) and showed very low homology to kinetoplast DNA (kDNA) and chromosomal DNA of Leishmania infantum and Leishmania tropica, two species commonly isolated from patients and wild hosts within foci of L. major in the Old World. DNA sequence analysis of a minicircle of L. major is reported for the first time, and it is demonstrated that this species shares with Leishmania aethiopica, Sauroleishmania tarentolae and several species of Trypanosoma a region of conserved sequence that is involved in DNA replication, a process that could present targets for selective chemotherapeutic attack. Sequence and restriction fragment analyses have indicated the difficulties of selecting species-specific sequences from kDNA which, even in the same parasite clone, contains several predominant minicircle classes, not all of which contain diagnostic sequences.

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences.

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.

Epidemiologic surveillance of visceral leishmaniasis in Sicily, Italy.

Visceral leishmaniasis (VL) is endemic in Sicily. Although it is a notifiable disease, there is evidence that the actual number of cases is higher than that reported. In 1987, a regional reference center for active surveillance of VL was established and it recorded a total of 284 cases through 1995, a mean of 31.5 cases/year and about four-fold more than previously reported. Of the 284 cases, 150 (53%) were children (< or = 14 years of age), and of the 134 adults, 39 (29%) were coinfected with human immunodeficiency virus (HIV). The commonest viscerotropic zymodeme of Leishmania infantum, MON 1, was identified in 40 (93%) of 43 HIV-negative and eight (57%) of 14 HIV-positive patients. Among 280 patients evaluated (i.e., all HIV-negative and 35 of 39 HIV-positive subjects), 254 (91%) were treated with meglumine antimoniate alone or in combination with other drugs; 23 (8%) received allopurinol or amphotericin B, either conventional or in liposomal form; and three terminally ill patients were not treated. Among the 245 HIV-negative patients, 236 (96%) were successfully cured, while nine (4%) (seven adults) died during the course of antimonial treatment. None of the 35 HIV-positive patients was definitively cured, although mortality was apparently associated with other opportunistic infections.

Studies on the prevalence of leishmanin skin test positivity in the Baringo District, Rift Valley, Kenya.

The leishmanin skin test (LST) was applied in 26 clusters of an average of 97 individuals in Baringo District, Kenya. These clusters were centered around recent cases of visceral leishmaniasis (VL). Of 2,411 individuals tested, 254 (10.5%, 155 males and 99 females) had a positive reaction. Among cured VL patients, the frequency was approximately 30% and no sex difference was observed. In the population as a whole, LST positivity increased with age to a stable level from approximately 15 years of age, reflecting an endemic situation. The level of LST positivity was 25-30% and 10-15% in males and females, respectively. Uninfected household contacts of VL cases had a higher frequency of LST reactivity than the rest of the population. This relationship was significant only in females and children, the prevalence ratio being 2.3 (95% confidence interval 1.3-4.1), 1.9 (1.1-3.5), and 1.4 (0.8-2.5) for females, children, and males, respectively. The frequency of LST positivity was higher individuals living in wood houses than in individuals living in house with mud or stone walls. Again, this difference was significant only in females and children (P = 0.02 and P = 0.04), but not in males (P = 0.7). The results suggest that children and women are exposed to the parasite in or around their houses, whereas adult males are, in addition, exposed elsewhere.

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy.

Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortment patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.

HIV-Leishmania co-infections in Italy: serological data as an indication of the sequence of acquisition of the two infections.

Twenty-two sera from visceral leishmaniasis (VL) patients with human immunodeficiency virus (HIV) infection (50% with the acquired immune deficiency syndrome) were examined for anti-Leishmania immunoglobulin G (IgG) antibodies and compared with 35 sera from VL patients without HIV (controls). Significant titres of specific IgG were found in 81.8% of co-infections. However, while control sera showed a restricted range of anti-Leishmania IgG titres, sera from co-infection cases displayed a considerable degree of variability, both quantitative and qualitative. They were clearly divided into 2 groups: one (18 sera) showing a continuous grading from nil to mid-concentrations of specific antibodies, the other (3 sera) showing titres 30-fold higher than this range. Taking into account the major immunological abnormalities involving humoral response described in HIV patient, the 2 groups may reflect a different sequence of acquisition of the 2 infective agents; the former representing VL acquired after HIV infection, and the latter representing the contrary situation.

Leishmania infantum sensu lato as an agent of cutaneous leishmaniasis in Abruzzi region (Italy).

The extreme difficulty of culturing in vitro parasites from Italian causes of cutaneous leishmaniasis (CL) has so far been a handicap to their biochemical identification. A technique based on the "transient growth" of promastigotes originating from parasites visceralized in LHC inbred hamsters was used. Through this technique, promastigote extracts of seven Leishmania isolates from CL cases from the Abruzzi region were obtained. Isoenzyme profiles of these isolates and reference strains of L. tropica, L. major and L. infantum were compared using 11 enzymes (ASAT, PGM, GPI, 6PGD, G6PD, ME, MDH, MPI, IDH, NH1 and NH2). Six isolates differed from L. infantum in one enzyme only (NH1), and one isolate was indistinguishable from L. infantum for all enzymes tested.

HIV-Leishmania co-infections in Italy. Isoenzyme characterization of Leishmania causing visceral leishmaniasis in HIV patients.

Visceral leishmaniasis (VL) infections in patients with human immunodeficiency virus (HIV) infection are dramatically increasing in Mediterranean countries such as Spain, France and Italy. A study has been carried out to characterize biochemically the agents of typical or unusual VL in subjects with HIV infection and to compare results with those obtained so far from VL and cutaneous leishmaniasis (CL) infections in HIV negative subjects. Twelve Leishmania stocks were isolated from 8 HIV patients and typed through the electrophoretic analysis of 14 isoenzymes. All the stocks were identified as L. infantum s.l. According to zymodeme classification, the results can be summarized as follows: (i) only half of the subjects were infected with the expected commonest viscerotropic zymodeme in the Mediterranean area, MON 1; (ii) 2 patients were infected with the most widespread agent of CL in Italy, L. infantum MON 24; (iii) one subject was found infected with a zymodeme (MON 78) which, so far, has been found only in Malta as an agent of CL; (iv) one subject was infected with a new zymodeme, MON 136, which shares biochemical characteristics with 2 dermotropic L. infantum zymodemes, MON 78 and MON 111. Thus, half of the HIV patients surveyed displayed severe visceralization of parasites usually showing low virulence in HIV negative subjects.

Natural infection of Phlebotomus perfiliewi with Leishmania infantum in a cutaneous leishmaniasis focus of the Abruzzi region, Italy.

One out of 213 Phlebotomus perfiliewi caught from an endemic cutaneous leishmaniasis focus in Abruzzi region (Italy) was naturally infected with promastigotes. The parasites were grown in culture medium and in a hamster, typed by the examination of 11 isoenzymes, and found to be indistinguishable from Leishmania infantum s.st. (Montpellier zymodeme 1). The probable role of P. perfiliewi in the transmission of cutaneous leishmaniasis due to L. infantum s.l. in the focus is discussed.