Luciana Fumarola

Anisakis pegreffi etiological agent of gastric infections in two Italian women.

Two cases of gastric anisakiasis have been documented in two Italian women who had consumed raw anchovies (Engraulis encrasicolus). The first patient was a 49-year-old woman presenting with epigastric pain and bloody vomiting after ingestion of marinated (vinegar) raw anchovies. During the esophagogastroduodenoscopy (EGDS) a white color worm was detected and extracted from cardia by means of biopsy forceps. The second patient was a 59-year-old woman with irritable bowel syndrome and gastritis, who underwent to periodical EGDSs. In the course of the last EGDS, a white color round worm on antrum and a small polyp on the fundus of the stomach were observed. The two nematodes have been identified as L3 larvae of the genus Anisakis by a light microscope, and as Anisakis pegreffi by polymerase chain reaction-restriction fragment length polymorphism. The molecular identification of the etiological agent at the species level allows to identify what Anisakidae species play a zoonotic role and which are the fish host species.

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.

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2.

Abstract Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-γ and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E2 has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E2. Experiments were also performed in the presence of the nitric oxide synthase inhibitor l-NGmonomethylarginine (l-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E2 exhibited increased nitric oxide producation and parasite killing, which were significantly reduced by either l-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E2. Taken together, these results indicate that prostaglandin E2 may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E2 on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E2 synthesis.

Isolation and Molecular Characterization of Free-Living Amoebae from Different Water Sources in Italy

Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.

Polymerase chain reaction detection of Bartonella henselae bacteraemia in an immunocompetent child with cat-scratch disease

Abstract A case of Bartonella henselae bacteraemia is reported in an immunocompetent 8-year-old boy with cat-scratch disease. Serology to B. henselae, diagnosed by polymerase chain reaction, was positive. DNA was extracted from peripheral whole blood and amplified with specific primers targeting the htrA gene of B. henselae. A non-isotopic hybridization assay with a species-specific oligonucleotide probe was used to detect the amplified product. Conclusion The polymerase chain reaction can be used for the rapid laboratory diagnosis of bacteraemia in cat-scratch disease.

Detection of Bartonella henselae and Afipia felis DNA by polymerase chain reaction in specimens from patients with cat scratch disease.

Abstract Polymerase chain reaction (PCR) amplification and colorimetric identification of amplicons were performed to detect Bartonella henselae and Afipia felis DNA in specimens from patients who were clinically and histologically suspected of having cat scratch disease. PCR products were revealed using 2% ethidium bromide agarose-gel electrophoresis and identified with specific probes in a commercial colorimetric hybridization assay (DEIA) (GEN-ETI-K; DiaSorin, Italy). Six paraffin-embedded lymph node biopsies from 18 patients as well as 18 samples of peripheral whole blood and 18 sera were investigated. Bartonella henselae DNA was recovered from the whole blood of four patients, and Bartonella henselae and Afipia felis DNA were detected in one patients lymph node biopsy. This study suggests that PCR-DEIA is sufficiently sensitive to be considered feasible for the molecular diagnosis of cat scratch disease.

Prevalence of antibodies to Bartonella henselae in patients with suspected cat scratch disease (CSD) in Italy

Cat scratch disease (CSD) is a relatively new diagnosed illness with clinical signs of self-limiting regional lymphadenopathy accompanied by symptoms of fever and malaise, to encephalopathy and neuropathy, occurring after a cat scratch or flea bite. Bartonella henselae is now accepted as the etiologic agent of CSD. From January 1994 to September 1998, 412 patients were evaluated for suspect CSD in Italy. Sera were tested for antibodies to B. henselae by a commercially available indirect immunofluorescent assay (IFA), based on B. henselae-infected Vero-cells as the antigen substrate. Of the 412 patients, 26 (6.3%) were considered positive having titers of immunoglobulin G (IgG) to B. henselae of 64 or higher. In these patients CSD was indeed confirmed by either histopathologic examination of lymph nodes biopsy or fourfold raise in antibody titers. Nevertheless, sera were tested by IFA for Afipia felis and one showed a double reactivity to B. henselae and A. felis. Finally, three sera, negative to B. henselae serology, were positive to A. felis. Three hundred and eighty-six patients received alternative diagnoses. One hundred and twenty-five serum samples from control subjects were negative by IFA for either B. henselae or A. felis. Moreover, a cross-reactivity with sera from patients affected by other diseases was not observed. Our study shows that the ascertained cases of CSD are etiologically determined by B. henselae, IFA assay is confirmed as a useful tool in the laboratory diagnosis and, over a 5 years period of study, the incidence of CSD in Italy has been low.

Helicobacter pylori Seroprevalence in Selected Groups of Albanian Volunteers

Background:Albania is a Mediterranean, South–East European developing country where epidemiological data on infectious diseases are scarce. In this study, the seroprevalence of Helicobacter pylori infection in 1,088 Albanian healthy volunteers (472 females followed-up to a prenatal clinic, 173 recruits, 443 health care workers) was evaluated.Materials and Methods:Sera were tested for immunoglobulin-G (IgG) antibodies against H. pylori using a quantitative enzyme immunosorbent assay.Results:The overall H. pylori seroprevalence was 70.7%. The H. pylori seroprevalence increased by age, from 60.4% in individuals younger than 20 years to 81% among those ‡ 50 years of age with a significant trend of increase by age. The overall seroprevalence was 73.9% for females and 59.5% for males. In addition the seroprevalence was 55.3% for people living in rural areas and 72.3% for people living in urban areas. No significant differences were found according to level of education except for individuals with elementary level of education. Nurses and hospital auxiliaries have significantly higher H. pylori seroprevalence when compared to other health care workers (physicians and office workers). When each variable (age, gender, area of residence, occupation, and education level) was adjusted for the confounding effect of the other variables by stepwise logistic analysis, we observed that age greater than 40 years and female gender remain the only variables independently associated with the presence of H. pylori IgG antibodies.Conclusions:H. pylori is highly prevalent among the Albanian population. Improving living conditions, education in hygiene, and the supply of running water are measures to prevent the transmission of H. pylori infection and other infections spread by the fecal–oral route in Albania.

The source of Helicobacter pylori infection in the neonatal period

Abstract Aims: To investigate the transmission of Helicobacter pylori in the perinatal period. Methods: H. pylori status of 180 women was preliminarily screened by serology and stool antigen test (SAT) within the fourth day after delivery and a positive value was confirmed by 13C-urea breath test. Infants were analyzed by SAT at 1, 6, 12 and 18 months of age. H. pylori status was also investigated in the medical and paramedical staff of the Neonatology Unit. Results: H. pylori infection was found in 34.4% of the women. At the 1st month of age, 5 out of 172 newborns (2.9%) were H. pylori positive by SAT. Three out of the five positive infants were born to H. pylori non-infected mothers. Formula feeding (P=0.02) and admission in intermediate-risk neonatal unit (P=0.01) were significantly related to a positive H. pylori result. Medical and paramedical staff of the neonatology unit was found H. pylori positive in 34.8% of cases. All five H. pylori positive children spontaneously cleared the infection. Conclusions: Perinatal H. pylori SAT positive status is low in our area, and it may be found both in infants from infected or non-infected mothers. Formula feeding and admission in intermediate-risk neonatal unit appear to be related with H. pylori infection.

Recombinant K39 immunochromatographic test for diagnosis of human leishmaniasis

A new recombinant K39 immunochromatographic test (ICT) was compared with the immunofluorescent antibody assay (IFA) for the rapid serological diagnosis of visceral leishmaniasis (VL) in Apulia, Southern Italy. A total of 264 individuals were tested, including 19 patients with VL (three of which were HIV positive), 67 individuals with suspected VL, 40 healthy controls and 138 patients with other diseases. The ICT was positive in all 19 patients with VL and negative in sera from the remaining individuals. Both the sensitivity and specificity of ICT was 100%. The ICT also worked well in HIV-Leishmania co-infected patients. Antibodies to Leishmania detected by the IFA and ICT remained at detectable levels for up to 12-24 months. A positive reaction by the ICT was detectable at a serum dilution of up to 1:20,480, indicating that a strong immunoresponse is mounted against the recombinant K39 antigen. In conclusion, the ICT is highly sensitive, specific, rapid, noninvasive and cost effective (euro8.43 for ICT and euro12 for IFA) in the diagnosis of VL in areas of low VL endemicity.