Marina Gramiccia

The northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors

Visceral leishmaniasis (VL) incidence has been increased in Italy in humans and dogs since the 1990s, with new foci being detected within traditional boundaries of endemic transmission but also in northern regions previously regarded as non‐endemic. To monitor the putative VL spreading, surveillance was implemented in northern continental Italy comprising: analysis of human cases recorded from 1990 through 2005; retrospective literature analysis of canine leishmaniasis (CanL) and phlebotomine sandfly records through 2002; prospective investigations in dogs from 2003 through 2005 and surveys on sandflies in 2003 and 2004. Two‐hundred‐thirty human cases (11% of Italian cases) were recorded. Their stratification by age and HIV status disclosed a sharp decrease of HIV/VL co‐infections paralleled by concomitant increase of paediatric and HIV‐negative adult patients during the study period. Four patients had no travel history. Seven leishmaniasis foci were retrospectively identified since 1990, whereas prospective investigations in dogs disclosed 47 autochthonous clinical cases and 106 autochthonous seropositives among 5442 dogs (2.1%) from 16 foci of six regions. Parasites were typed as Leishmania infantum MON‐1. Four vector species were identified among 1696 Phlebotomus (Larroussius) collected specimens. Comparisons with historical data showed that P. perniciosus and P. neglectus have increased in density and expanded their geographic range in the study area. Northern continental Italy is now focally endemic for VL and a moderate risk for human disease does exist, although the intensity of transmission seems to be lower than in traditional settings of Mediterranean VL.

Studies on canine leishmaniasis control. 1. Evolution of infection of different clinical forms of canine leishmaniasis following antimonial treatment

81 dogs naturally infected with Leishmania infantum in the Isle of Elba, Italy, were treated with meglumine antimoniate (Glucantime). 36 of them (45.5%) were asymptomatic cases. 4-24 months after treatment the dogs were clinically and serologically examined; the recovery rates were 47.2% for asymptomatic cases, 33.3% for oligosymptomatic cases, and 11.1% for symptomatic cases. Furthermore, treatment had prevented the development of patent disease in 90% of non-recovered asymptomatic cases, whereas it had produced only slight improvement of clinical condition in patent dogs which were still infected after drug administration. Treatment with antimonial drugs is therefore recommended in canine leishmaniasis control if non-patent or sub-clinical forms of the disease are detected by seroepidemiological surveys.

Incidence and Time Course of Leishmania infantum Infections Examined by Parasitological, Serologic, and Nested-PCR Techniques in a Cohort of Naïve Dogs Exposed to Three Consecutive Transmission Seasons

ABSTRACT Most experience in the comparison of diagnostic tools for canine leishmaniasis comes from cross-sectional surveys of dogs of different ages and breeds and in cases with unknown onset and duration of leishmaniasis. A longitudinal study was performed on 43 beagle dogs exposed to three transmission seasons (2002 to 2004) of Mediterranean leishmaniasis and examined periodically over 32 months through bone marrow microscopy and nested PCR (n-PCR), lymph node culture, serology (immunofluorescent-antibody test), and evaluation of clinical parameters. Starting from January 2003, the highest rate of positives was detected by n-PCR at all assessments (from 23.3% to 97.3%). Sensitivities of serologic and parasitological techniques were lower but increased with time, from 15.8% to 75.0 to 77.8%. Some dogs that tested positive by n-PCR but negative by other tests (“subpatent infection”) remained so until the end of the study or converted to negative in subsequent assessments, whereas all dogs with positive serology and/or microscopy/culture (“asymptomatic patent infection”) exhibited progressive leishmaniasis; 68% of them developed clinical disease (“symptomatic patent infection”) during the study, at 7 (range, 3 to 14) months after being positive to all tests. Postexposure infection incidences were high and were significantly different between 2002 and 2003 exposures (39.5% and 91.7%, respectively). The time course of infection was highly variable in each dog, with three patterns being identified: (i) rapid establishment of a patent condition (0 to 2 months from detection of infection); (ii) a prolonged subpatent condition (4 to 22 months) before progression; and (iii) a transient subpatent condition followed by 10 to 21 months of apparent Leishmania-negative status before progression.

Clinical Use of Polymerase Chain Reaction Performed on Peripheral Blood and Bone Marrow Samples for the Diagnosis and Monitoring of Visceral Leishmaniasis in HIV-Infected and HIV-Uninfected Patients : A Single-Center, 8-Year Experience in Italy and Review of the Literature

BACKGROUND To overcome some of the limitations of conventional microbiologic techniques, polymerase chain reaction (PCR)-based assays are proposed as useful tools for the diagnosis of visceral leishmaniasis. PATIENTS AND METHODS A comparative study using conventional microbiologic techniques (i.e., serologic testing, microscopic examination, and culture) and a Leishmania species-specific PCR assay, using peripheral blood and bone marrow aspirate samples as templates, was conducted during an 8-year period. The study cohort consisted of 594 Italian immunocompetent (adult and pediatric) and immunocompromised (adult) patients experiencing febrile syndromes associated with hematologic alterations and/or hepatosplenomegaly. Identification of the infecting protozoa at the species level was directly obtained by PCR of peripheral blood samples, followed by restriction fragment-length polymorphism analysis of the amplified products, and the results were compared with those of isoenzyme typing of Leishmania species strains from patients, which were isolated in vitro. RESULTS Sixty-eight patients (11.4%) had a confirmed diagnosis of visceral leishmaniasis. Eleven cases were observed in human immunodeficiency virus (HIV)-uninfected adults, 20 cases were observed in HIV-infected adults, and the remaining 37 cases were diagnosed in HIV-uninfected children. In the diagnosis of primary visceral leishmaniasis, the sensitivities of the Leishmania species-specific PCR were 95.7% for bone marrow aspirate samples and 98.5% for peripheral blood samples versus sensitivities of 76.2%, 85.5%, and 90.2% for bone marrow aspirate isolation, serologic testing, and microscopic examination of bone marrow biopsy specimens, respectively. None of 229 healthy blood donors or 25 patients with imported malaria who were used as negative control subjects had PCR results positive for Leishmania species in peripheral blood samples (i.e., specificity of Leishmania species-specific PCR, 100%). PCR and restriction fragment-length polymorphism analysis for Leishmania species identification revealed 100% concordance with isoenzyme typing in the 19 patients for whom the latter data were available. CONCLUSIONS PCR assay is a highly sensitive and specific tool for the diagnosis of visceral leishmaniasis in both immunocompetent and immunocompromised patients and can be reliably used for rapid parasite identification at the species level.

Feline leishmaniosis due to Leishmania infantum in Italy

A case of leishmaniosis in domestic cats (Felis catus domesticus) is described. The subject showed a nodular lesion on the eyelid. The diagnosis was achieved by serological, parasitological, and light and electron microscopic investigations. By molecular techniques the aetiological agent was identified as belonging to Leishmania infantum, the species implicated in human and canine leishmaniosis in southern Europe. A preliminary study on the prevalence of asymptomatic feline leishmaniosis, performed in the areas where the infected cat was identified, revealed a low seroprevalence of infection: only 1 (0.9%) of the 110 cat sera examined by indirect fluorescent antibody test was positive for anti-Leishmania antibodies. Because clinical signs in feline leishmaniosis are unspecific and similar to those observed in other diseases commonly found in this species, leishmaniosis must be added to the differential diagnosis by feline veterinary practitioners and adequate serologic and histopathologic investigations must be performed in endemic areas.

Decreased sensitivity to meglumine antimoniate (Glucantime) of Leishmania infantum isolated from dogs after several courses of drug treatment

Although unresponsiveness to antimonial drugs in human leishmaniasis appears to be increasing, resistance to antimony in Leishmania is not well documented. Treatment of leishmaniasis in dogs, the domestic reservoir of L. infantum, with meglumine antimoniate (Glucantime) is a common practice in many Mediterranean countries. The dogs, however, remain highly infective to the phlebotomine vectors, even after several courses of treatment. A study was therefore carried out to test the comparative susceptibility to meglumine antimoniate of L. infantum stocks isolated from four naturally-infected dogs, before (BT) and after treatment (AT) with three to six courses of the drug, and used to infect Balb/c mice. Significant differences in suppression between the BT and AT stocks were observed in the infected mice when they were given the drug at a rate of 0.01-10 mg kg-1 day-1 for five days. Each AT stock was between eight and 41 times more resistant to meglumine antimoniate than the BT stock from the same dog, in terms of the ratios of the AT ED50 values to the corresponding BT values, which were calculated as indices of resistance. This result underlines the futility and danger of repeated antimonial treatments of dogs with signs of leishmaniasis, as these may produce a permanent reservoir of parasites unsusceptible to the drugs in human clinical use.

Role of PCR in Diagnosis and Prognosis of Visceral Leishmaniasis in Patients Coinfected with Human Immunodeficiency Virus Type 1

ABSTRACT A group of 76 consecutive human immunodeficiency virus (HIV)-positive patients with fever of unknown origin (n = 52) or fever associated with pulmonary diseases was evaluated in order to assess the usefulness of PCR with peripheral blood in the diagnosis and follow-up of visceral leishmaniasis. We identified 10 cases of visceral leishmaniasis among the 52 patients with fever of unknown origin. At the time of diagnosis, all were parasitemic by PCR with peripheral blood. During follow-up, a progressive decline in parasitemia was observed under therapy, and all patients became PCR negative after a median of 5 weeks (range, 6 to 21 weeks). However, in eight of nine patients monitored for a median period of 88 weeks (range, 33 to 110 weeks), visceral leishmaniasis relapsed, with positive results by PCR with peripheral blood reappearing 1 to 2 weeks before the clinical onset of disease. Eight Leishmania infantum and two Leishmania donovani infections were identified by PCR-restriction fragment length polymorphism analysis. PCR with peripheral blood is a reliable method for diagnosis of visceral leishmaniasis in HIV-infected patients. During follow-up, it substantially reduces the need for traditional invasive tests to assess parasitological response, while a positive PCR result is predictive of clinical relapse.

Epidemiological surveillance of leishmaniasis in Hiv-1-infected individuals in Italy

ObjectiveTo 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. DesignRetrospective and prospective surveillance study. PatientsItalian patients with HIV-1 infection and leishmaniasis diagnosed between 1985 and 1994. ResultsWe 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. ConclusionsActive 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.

Leishmaniases in the Mediterranean in the era of molecular epidemiology

Molecular tools are used increasingly for descriptive epidemiological studies in different Mediterranean foci of visceral and cutaneous leishmaniases. Several molecular markers with different resolution levels have been developed to address key epidemiological questions related to the (re-)emergence and spread of leishmaniases, as well as its risk factors: environmental changes, immunosuppression and treatment failure. Typing and analytical tools are improving but are not yet addressing all epidemiological issues satisfactorily. There is an urgent need for better cooperation between laboratory scientists and epidemiologists and for regional epidemiological surveillance of these infectious diseases that affect all Mediterranean countries.

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.