Olga Brandonisio

Effect of Antiretroviral Therapy on Cryptosporidiosis and Microsporidiosis in Patients Infected with Human Immunodeficiency Virus Type 1

Abstract To better understand whether potent antiretroviral therapies can modify the natural history of HIV-1-associated microsporidiosis and cryptosporidiosis, the response to antimicrobial treatment of these opportunistic infections was evaluated in patients with or without antiretroviral treatment. Fifty patients with diarrhoea, all positive for Cryptosporidium parvum or Enterocytozoon bieneusi, were included in the study. Retrospective data were collected concerning demographics, clinical and microbiological characteristics of the parasitic infection, antiretroviral therapy and prophylaxis against opportunistic infections. Faecal samples were prepared using the Richie formalin-ethyl acetate method and stained using the modified Ziehl-Neelsen method for detection of Cryptosporidium parvum and Isospora belli, the modified trichrome and calcofluor white technique for detection of Enterocytozoon spp., and iodine for detection of ova, cysts or vegetative forms. Diarrhoea was defined as an abnormal increase in stool liquidity, an abnormal increase in stool frequency and a daily stool weight of more than 250 g for a period of at least 4 days. Patients treated with double antiretroviral therapy or protease inhibitors demonstrated an excellent response and a sustained therapeutic effect after follow-up (range, 5–36 months). The relapse of cryptosporidiosis in two patients who discontinued antiretroviral therapy suggests that the infection might remain in a latent stage. The resolution of the diarrhoea seems to be related to an increased CD4+ cell count rather than to the viral load. In conclusion, these data strongly support the hypothesis that combination antiretroviral therapy is able to greatly modify the course of cryptosporidiosis and microsporidiosis in patients infected with HIV-1.

Giardia and Cryptosporidium and public health: the epidemiological scenario from the Italian perspective.

Giardia and Cryptosporidium spp. are protozoa that cause human and animal disease worldwide and often exhibit zoonotic transmission. This review gives ample information concerning the epidemiology of these parasites in Italy, i.e. prevalence data in humans, farm and pet animals, shellfish and aquatic environment. Moreover, it reports genotyping results obtained from different isolates, with particular emphasis on the spread of host-specific and zoonotic species/genotypes of various origin, and on molecular data that make the Italian situation different from that of other countries. Finally, possible explanations are given for the infrequent reports of Giardia and Cryptosporidium spp. outbreaks, despite widespread faecal contamination by these parasites.

Canine leishmaniasis in the Gargano promontory (Apulia, South Italy)

We examined 444 dogs by visiting farms and peripheral districts on the Gargano promontory (Province of Foggia, Apulia, South Italy). Clinical examination and immunofluorescence antibody test for leishmaniasis were performed. Bone marrow and lymph node samples obtained from 25 dogs with positive serological test were cultured on Tobie-Evans medium. The results obtained show both a higher seropositivity rate for canine leishmaniasis (14.4%) and a higher percentage of asymptomatic dogs with positive serological test (53.1%) compared to previous research in the same area. Seven strains isolated from infected dogs belonged to Leishmania infantum species zymodeme Montpellier 1. None of 82 sera of humans living in close contact with infected dogs were positive for leishmaniasis.

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1α induce nitric oxide release and enhance parasite killing in leishmania infantum-infected human macrophages

Abstract. Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-NGmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.

Nitric oxide production by macrophages of dogs vaccinated with killed Leishmania infantum promastigotes

Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.

Giardia cysts and Cryptosporidium oocysts in membrane-filtered municipal wastewater used for irrigation

ABSTRACT A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.

Infection with Leishmania infantum Inhibits Actinomycin D‐Induced Apoptosis of Human Monocytic Cell Line U‐937

Abstract. Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D‐induced apoptosis of the human monocytic cell line U‐937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite‐free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis‐inducing agent. Actinomycin D‐induced apoptosis in U‐937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U‐937 cells via a mechanism involving inhibition of caspase‐3 activation. Furthermore, protein kinase C δ (PKC δ) cleavage was increased in actinomycin D‐treated U‐937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC‐mediated pathways by LPG can be implicated in the enhanced survival of the parasites.

Evaluation of a Rapid Immunochromatographic Test for Serodiagnosis of Visceral Leishmaniasis

Abstract.The purpose of this study was to compare the performance of a rapid immunochromatographic dipstick test for the qualitative detection of circulating antibodies to the leishmanial recombinant antigen K39 with that of a classical immunofluorescent antibody test for serodiagnosis of visceral leishmaniasis. Sera from 143 Italian subjects, including 69 patients with clinically suspected visceral leishmaniasis, 23 patients with hypergammaglobulinemia and 51 healthy controls, were tested. The immunochromatographic test was performed according to the manufacturers instructions, using antigen-impregnated nitrocellulose paper strips. The immunofluorescent antibody test was performed according to an established method, using promastigotes of Leishmania infantum zymodeme Montpellier 1 as antigen. In 11 patients, diagnosis of active Leishmania infection was established by microscopic examination of biopsy samples and/or clinical response to meglumine antimoniate. Results of the two tests correlated for all but two sera examined. In two patients, one with proven infectious mononucleosis and one with bacterial pneumonia, the immunofluorescent antibody test was positive and the dipstick test was negative. In the restricted sample of patients in whom a definitive diagnosis was established, the immunochromatographic test was positive in 11 of 11 patients with confirmed Leishmania infection and negative in 103 of 103 subjects who either had other documented diseases or were healthy controls, showing 100% sensitivity and 100% specificity.

Evidences for iNOS expression and nitric oxide production in the human macrophages.

Nitric oxide (NO) is a pleiotropic mediator of numerous biological processes, including smooth muscle relaxation, neurotransmission and defence against pathogens. In addition, NO is involved in the pathogenesis and control of inflammation, tumors, autoimmunity, and infectious and chronic degenerative diseases. NO, a highly reactive radical, is produced from L-arginine and oxygen by the enzyme NO synthase (NOS). Three NOS isoforms have been identified: two distinct NOS isoforms are constitutively expressed in cells, whereas a third isoform, inducible NOS (iNOS), is transcribed in response to specific stimuli. In particular, iNOS is responsible for the discontinuous synthesis of high amounts of NO and was originally characterized in murine macrophages after exposure to cytokines and/or microbial products. A wide range of microorganisms is sensibly inhibited in its development by NO, like fungi, bacteria, protozoa and viruses. Although NO production and its antimicrobial effect appear well established in rodent macrophages, the existence of L-arginine pathway in human mononuclear phagocytes has long been disputed. Recently, evidences showing the iNOS activity and NO production in other animal models, including humans, are now emerging, even if the NO induction has been more difficult to demonstrate. The present observations provide evidence for the occurrence of iNOS protein expression and NO production in human macrophages cultured in vitro.

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy.

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.