Lorenzo Ciceroni

Epidemiological trend of human leptospirosis in Italy between 1994 and 1996

In the three-year period 1994–1996, 222 reports on human cases of leptospirosis were received by the Italian Ministry of Health. The average annual number of reports was 29.2% lower than in the preceding eight years. In all cases but two the infections were thought to have been acquired in Italy. As in previous years, the majority of cases was observed in the northern regions of the country (83.8%), mostly in males (88.9%). Cases occurred in all age groups, but were more common in the working-age population (15–64 years). There was no common-source outbreaks. The typical leptospiral seasonal course, with a peak in August, was observed. During 1994, leptospirosis was the reported cause of death in 19 patients. Mortality was higher among males than females. The overall fatality rate was 22.6%. During the study period, a total of 126 cases of leptospirosis were confirmed by the National Centre for Leptospirosis or one of the 12 Regional Leptospira Laboratories. Of the 103 patients for whom information on place of residence, contact with animals, occupational and recreational activities was available, 98 (95.1%) were people who live in rural areas or devote themselves to occupational or recreational activities at risk. The likely source of infection and the mode of exposure were known for 55 patients. Forty-five patients (81.8%) were likely infected by contaminating water (43 cases) or soil (2 cases), ten (18.2%) by direct contact with animals or animal urine. Both running (51.2%) and stagnant water (27.9%) have been reported as a source of infection. Rodents were implicated in 50.0% of the 10 cases involving animals. In comparison with the preceding eight-year period, the risk of contracting leptospirosis was found to have increased for recreational activities (from 34.7 to 38.2%) and decreased for occupational activities (from 45.8 to 32.7%). A large number of infections, however, was ascribed to accidental events (25.5%). As in the previous period, besides fever, the involvement of the liver was the most frequent clinical manifestation (70.8%). Influenza-like symptoms were the only signs of illness in 15.1% of cases. Infections by 9 different serogroups were detected. The most frequent antibodies were those against serovars icterohaemorrhagiae, poi, copenhageni and bratislava. The presence of co-agglutinins against serovars belonging to different serogroups prevented the identification of the presumptive infecting serogroup in 19.8% of subjects.

Survey on the prevalence of leptospira infections in the Italian population

This investigation is the first nationwide survey on the circulation of leptospira infections in human beings in Italy. In nine out of twenty Italian regions, representative samples of the population were investigated for the presence of leptospira infections. Unexpectedly, leptospira infections were found to be widespread, the number of cases being much higher than the diagnosed clinical cases. There were found to be high, medium, and low risk areas. On the whole, the risk for the rural population was no higher than the risk for urban dwellers; leisure activities, contact with animals and residence on the plain versus residence in the hills were important risk factors. There was an unidentified risk factor in urbanites which was absent in the rural population. A changing pattern in infecting serovars was observed, with infections from serogroups Sejroe, Javanica and Australis prevailing over infections from the Icterohaemorrhagiae and Bataviae serogroups, which were the main agents of human leptospirosis during the 1950s. The mechanisms of these changes, the need for epidemiological surveys and improved diagnostic methods of screening are discussed.

Prevalence and incidence of antibodies to Borrelia burgdorferi and to tick-borne encephalitis virus in agricultural and forestry workers from Tuscany, Italy.

The ticks Ixodes persulcatus and Ixodes ricinus are the main vectors of both Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus in Eurasia. Borrelia burgdorferi is the cause of Lyme borreliosis, and TBE is a biphasic meningoencephalitis induced by an arbovirus belonging to the flavivirus family. The principal aims of the current investigation were (i) to determine the frequency of serological evidence of Borrelia burgdorferi sensu lato and TBE infections in healthy agricultural and forestry workers, (ii) to determine the incidence of seroconversion for antibodies against Borrelia burgdorferi sensu lato and TBE virus in Tuscan workers during a 1-year survey; and (iii) to assess the occupational risk for agricultural and forestry activities in a defined area (Tuscany, Italy). A total of 412 blood samples were taken from agricultural and forestry workers, and information on age, duration of employment, and history of tick bites was collected in a questionnaire to establish the risk factors for the diseases. Three hundred sixty-five blood donors from the same region served as controls. To estimate the rate of seroconversion, 176 of the agricultural and forestry workers were tested 1 year later. IgG and IgM antibodies against Borrelia burgdorferi sensu lato and TBE virus were detected in serum by an enzyme-linked immunosorbent assay and confirmed by Western blot analysis for Borrelia burgdorferi and by a test for inhibition of hemagglutination for TBE. Antibodies against Borrelia burgdorferi were more frequent among the workers than in the control group (7.8% vs. 4.9% in the IgG-IgM enzyme-linked immunosorbent assay and 7.03% vs. 3.56% in the confirmatory test). No seropositivity was observed for TBE virus. Eighteen of 176 subjects who underwent a second blood test developed specific antibodies against Borrelia burgdorferi within 1 year.

Current Knowledge of Rickettsial Diseases in Italy

Abstract:  Rickettsial diseases continue to be the cause of serious health problems in Italy. From 1998 to 2002, 4,604 clinical cases were reported, with 33 deaths in the period from 1998 to 2001. Almost all the cases reported in Italy are cases of Mediterranean spotted fever (MSF). Other rickettsioses that have been historically documented are murine typhus and epidemic typhus. Since 1950, only sporadic cases of murine typhus have been reported, and Italy currently appears to be free of epidemic typhus. As in other European countries, imported cases of rickettsialpox, African tick‐bite fever (ATBF), and scrub typhus have been reported. In 2004, three cases of a mild form of rickettsiosis were serologically attributed to Rickettsia helvetica.

Isolation and Characterization of Borrelia burgdorferi Sensu Lato Strains in an Area of Italy Where Lyme Borreliosis Is Endemic

ABSTRACT Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to “San Martino” Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii andB. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.

Human leptospirosis in Italy, 1986–1993

In the eight-year period 1986–1993, the Italian National Center for Leptospirosis and the Regional Leptospira Laboratories confirmed 312 cases of clinical leptospirosis by using the microscopic agglutination (MA) assay. The majority of cases was observed in Northern regions of the Country. Cases were reported in all age groups, but were most common in the working-age population. Of 312 cases, 291 (93.3%) occurred among males. The largest number of infections was ascribed to occupational activities (45.8%). The typical leptospiral seasonal course, with a peak during the summer, was observed. Involvement of the liver was the most frequent manifestation. Influenza-like symptoms were the only signs of illness in 11.1% of cases. Anti-leptospira antibodies, cross-reacting with two or more serovars, were found in 28.2% of sera. The most frequent serovar-specific antibodies were those againstpoi, icterohaemorrhagiae, bratislava, copenhageni andsejroe.

Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.

Recent trends in human leptospirosis in Italy

The recent epidemiological trends of human leptospirosis in Italy were investigated using data collected for the years 1981–1985. A total of 626 hospitalized patients with clinical diagnoses of suspected leptospirosis were reported by hospital centers from several Italian regions. Epidemiological, clinical and seroimmunological data were collected in 517 of these cases and examined by the National Center for Leptospirosis.Serological findings in 33.5% of these subjects met the criteria for confirmation of the disease. In 21.8% of the subjects, low titer antibodies were detected, which possibly reflected previous leptospiral infections. An early antibiotic treatment of the current infection may also have lowered the seroimmunological response in some of these patients.In 59.3% of the confirmed cases, modes of transmission were allotted equally between accidental events and recreational or occupational activities. Drinking water from an open air fountain emerged as an uncommon mode of transmission; it was responsible for an outbreak of 33 cases of leptospirosis. In another 37.07% of the subjects, it was impossible to establish the mode of transmission.Respiratory or influenza-like symptoms were the only clinical signs of illness in 21.2% of the patients with confirmed leptospirosis.In comparison to the sixties and seventies, the prevalence of infecting serovars showed increasing incidence of infections due to serovars of the Javanica (11.0%) and Australis (11.0%) serogroups and an important decrease in the Bataviae serogroup infections (from 58.8% in rice-field workers in the forties to 0.6% in the years 1981–1985). Sejroe serogroup infections accounted for 4.5 per cent of confirmed cases of leptospirosis.In 49.7% of subjects with confirmed leptospirosis, cross-agglutination at the same titre with two or more serovars of different sero-groups occurred, thus preventing the identification of the serogroup of the infecting strain.

Fatty Acid Profiles, a Chemotaxonomic Key for the Classification of Strains of the Family Leptospiraceae

The fatty acid profiles of 59 type strains representing genera, species, and serovars belonging to the family Leptospiraceae were investigated by using gas-liquid chromatography of fatty acid methyl ester (FAME) derivatives prepared from washed leptospires. The interstrain differences of the gas-liquid chromatography FAME profiles were quantified by performing a linear regression analysis in which we compared the FAME profiles of pairs of strains and expressed the results as correlation coefficients. Leptospiral strains could be differentiated into 17 FAME relatedness groups, which were characterized by both stringent similarities within a group and sharp differences between groups. In each group, a strain was selected as a FAME reference strain. FAME reference strains can be used as keys for a chemotaxonomic phenotypic classification of strains belonging to the family Leptospiraceae.

Prevalence of leptospirosis in man. Pilot survey

A survey on the prevalence of leptospirosis was performed on the population living in an area of central Italy. The size of the sample was calculated in order to provide significant results in the case of a prevalence of infection in not less than 1 % of the population.Results demonstrated an unexpectedly wide circulation of leptospirosis in the surveyed area, showing a prevalence rate of infection of 11.34% for people living in rural areas and 3:08% for people living in the main town. The highest prevalence of infection (17.44%) was found in people between 30 and 44 years of age, living in rural areas.Such a wide circulation of undiagnosed past leptospiral infections was attributed both to the prevalence of mild clinical cases of leptospirosis in humans and the lack of microbiological tests performed to differentiate current leptospirosis from other infectious illnesses. An unexpected persistence in sera of co-agglutinins towards non-pathogenic serovars of L. biflexa was also noticed in healthy people.Criteria were established for the extension of the survey on the prevalence of leptospirosis to cover larger areas by limiting sampling to the more exposed age groups and to areas representative of a larger land belt.