Donato Fumarola

Lyme disease in Italy.

We present the first cases of Lyme disease found in Italy. The diagnosis was based on clinical and laboratory data. The antigen used for indirect immunofluorescence (I.I.F.) was kindly supplied to us by Prof. R. Ackermann (Köln). Reciprocal titer was 64 in five patients, 128 in six, 256 in three and 512 in one. The patients came either from the Eastern Ligurian Coast or the Trieste Karst: these are consequently the first two Italian areas where Lyme disease has been recognized up to the present.

Downregulation of Human Polymorphonuclear Cell Activities Exerted by Microorganisms Belonging to the α-2 Subgroup of Proteobacteria (Afipia Fs and Rochallmaea Henselae)

Intracellular pathogens have evolved effective mechanisms in order to survive in an intracellular environment, thus avoiding destruction by phagocytic cells. In this regard, a correlation between resistance to phagocytic killing and expression of pathogenic potency has been established. In this report, we have studied the interaction between human polymorphonuclear cells (PMN) and two gram-negative microorganisms, Afipia felis and Rochalimaea henselae, which belong to the alpha-2 subgroup of the class Proteobacteria. A. falis has been previously proposed as the causative agent of Cat Scratch Disease (CSD), but several recent lines of evidence attribute a major role to R. henselae. Of note, CSD is a syndrome characterized by a chronic lymphoadenopathy, involving macrophages and endothelial cells with a progression towards a granulomatous process and/or angiogenesis. Since members of the alpha-2 subgroup of Proteobacteria have the property to survive intracellularly, we have evaluated the effects exerted by A. felis and R. henselae on human PMN in terms of chemotaxis locomotion, degranulation and oxidative metabolism. Results will show an impairment of PMN activities as a consequence of the challenge with both microrganisms. In particular, inhibition of PMN oxidative function occurred either as result of a direct exposure to both A. felis and R. henselae or when PMN were primed by bacteria for the N-formyl-methionyl-leucyl-phenylalanine enhancement of the oxidative burst. These findings may account for the ability of A. felis and R. henselae to survive within PMN as expression of a further mechanism of pathogenic potency, influencing also the nature and the evolution of inflammatory response in the lesion sites.

Clinical experience in detecting endotoxemia with the limulus test in typhoid fever and other Salmonella infections.

SummarySemiquantitative estimates of circulating endotoxin were performed by the limulus test in patients suffering from typhoid fever and other salmonelloses. The test was positive in a large number of cases. However, no clearcut correlation was found between existence of endotoxemia, as such, and pyrexia. A correlation with recent bacteremia was found for highest levels of endotoxin activity. In minor salmonelloses a striking prevalence of positive cases was observed in the age group under one year. These findings were discussed in relation to the diagnostic and pathogenetic facets of the problem.ZusammenfassungAn Patienten mit Typhus und anderen Salmonellosen wurden mit Hilfe des Limulus-Tests semiquantitative Bestimmungen des zirkulierenden Endotoxins vorgenommen. Der Test erwies sich in einer großen Zahl von Fällen als positiv. Es fand sich jedoch keine eindeutige Wechselbeziehung zwischen dem Vorhandensein von Endotoxämie als solcher und Pyrexie. Eine Wechselbeziehung zu Bakteriämie wurde nur bei höchsten Werten der Endotoxinaktivität festgestellt. Bei den leichteren Salmonellosen wurde ein auffallendes Überwiegen positiver Fälle in der Altersgruppe unter einem Jahr beobachtet. Diese Befunde wurden im Zusammenhang mit den diagnostischen und pathogenetischen Seiten des Problems diskutiert.

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.

Endotoxins, cytokines, and neuroimmune networks with special reference to HIV infection.

The mutual interplay between the nervous system and the immune system has been the object of extensive studies in either conditions of stress or human diseases such as multiple sclerosis, psychiatric disorders, and migraine.’” Initial studies by Selye4 demonstrated activation of the hypothalamus-pituitary-adrenal axis in normal animals in response to stressful events; these effects were similar to the wasting syndrome that follows the experimental administration of bacterial endotoxins or lipopolysaccharides

Endotoxin-like activity associated with lyme disease Borrelia

The newly recognized spirochete, Borrelia burgdorferi, the causative agent of Lyme Disease, has been examined for endotoxin-like activities as measured by the standard Farmacopea Ufficiale della Republica Italiana rabbit fever test and the Limulus amoebocyte lysate assay. The suspension of heat-killed microorganism caused a febrile response at a dose of 1 X 10(8) bacteria pro kilo. Similar results were obtained in the Limulus assay where the heat-killed spirochetes stimulated formation of solid clot until the concentration of 1 X 10(5) per ml. Both in pyrogen test and in Limulus assay heat-killed Escherichia coli exhibited a higher degree of potency. These results show that LD-Borrelia possess endotoxin-like activities which could help in understanding the pathogenesis of the clinical symptomatology of the disease.

In vitro production of tumor necrosis factor-α, interleukin-6 and interleukin-8 from normal human peripheral blood mononuclear cells stimulated by Rhodococcus equi

The capability of heat-killed Rhodococcus equi organisms to induce in vitro release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8 from normal human mononuclear cells as well as the secretion kinetics of these inflammatory cytokines over a 48 h period were evaluated. Results show that normal human mononuclear cells are efficiently triggered to secrete TNF-alpha, IL-6 and IL-8 following R. equi stimulation according to a different kinetics. In particular, release of IL-B was already maximally expressed after 2 h of stimulation, while TNF-alpha amounts progressively increased in a time-dependent fashion. Finally, IL-6 secretion reached peak levels as soon as 18 h of incubation. Taken together, these data point out that monocyte-derived cytokines may play an important role in the immunological control of R. equi infection in immunocompetent people.

Adherence of lyme disease spirochetes to rat lymphocytes

In the present work, the capacity of Lyme disease (LD) Spirochetes to spontaneously adhere to rat lymphocytes has been evaluated. Borrelia organisms adhere to thymus, spleen, Payers patches and peripheral blood lymphocytes in a higher frequency than that observed with Salmonella minnesota R345 (Rb) used as reference strain. Employing enriched splenic T and B cell populations, Spirochete binding to B lymphocytes is more elevated than that obtained with Salmonella, while similar percentage of T cells forms rosettes with both microorganisms. Taken together, these findings provide evidence for a novel mode of interaction between LD Spirochetes and host immune system.