Bacteriology
Presence of anti-Borrelia burgdorferi antibodies and Borrelia burgdorferi sensu lato DNA in samples of subjects in an area of the Northern Italy in the period 2002–2008

https://doi.org/10.1016/j.diagmicrobio.2010.09.007Get rights and content

Abstract

Lyme borreliosis (LB) is a multisystemic disease caused by Borrelia burgdorferi sensu lato complex transmitted to humans by Ixodes ticks and whose epidemiology is poorly investigated in Europe. In this study an epidemiologic survey on the prevalence of the causative agent of such infectious disease was performed in the area of Parma (Northern Italy) during 2002–2008. Serum samples belonging to 2336 patients and cerebrospinal fluid samples (belonging to 42 of the same patients) were analyzed for serologic diagnosis of LB. Direct laboratory assays [polymerase chain reaction (PCR) and cultivation] were performed on samples belonging to patients with the clinical suspicion of LB. The seroprevalence was 0.55% considering the subjects with both anti-B. burgdorferi IgG and IgM. The samples tested by culture and PCR were all negative except for a tick removed from the skin of a healthy man. The results suggest that infection by B. burgdorferi in this area quite rarely occurs.

Introduction

Lyme borreliosis (LB) is a multisystemic disease caused by spirochetes belonging to Borrelia burgdorferi sensu lato complex that is sustained mainly by wild animals and transmitted to humans by ticks belonging to the genus Ixodes. The main pathogenic genomic species responsible for human disease in Europe are Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto included in the B. burgdorferi sensu lato complex. B. burgdorferi sensu stricto is the sole species causing human infection in the United States (Aguero-Rosenfeld et al., 2005).

The spread of LB depends on geographical, environmental, and climatic factors. Epidemiologic data about LB mostly derive from local/regional surveys. In Europe it has become an important infectious disease, most prevalent in forested areas such as Scandinavia and Central Europe (Franz and Krause, 2003).

As concerns the diagnosis, besides the clinical evaluation, different approaches have been used in the clinical laboratory: principally direct assays, such as culture in the Barbour, Stoenner and Kelly medium II (BSK-II) (Sigma Aldrich, Milwaukee, WI) and detection of B. burgdorferi-specific nucleic acid, and indirect assays based on specific antibodies detection (Aguero-Rosenfeld et al., 2005). Among these, culture undoubtedly offers the best confirmation of an active infection (Aguero-Rosenfeld et al., 2005). At the same time, culture of B. burgdorferi has a low recovery rate and may require many weeks before growth of the bacteria is evident (Exner and Lewinski, 2003).

The first sign of infection is usually a circular rash called erythema migrans (EM). Patients also experience symptoms of fatigue, chills, fever, headache, muscle and joint aches, and swollen lymph nodes. Untreated, the infection may spread to other parts of the body within a few days to weeks, producing an array of discrete symptoms (including loss of facial muscle tone called “Bell's palsy”, severe headaches, hearth palpitations, and pain that moves from joint to joint). After several months, approximately 60% of patients with untreated infection will begin to have intermittent bouts of arthritis (involving large joints), with severe joint pain and swelling. In addition, up to 5% of untreated patients may develop chronic neurologic complaints months to years after infection (www.cdc.gov).

While Lyme arthritis is the most common late manifestation of LB in North America, acrodermatitis chronica atrophicans appears to be the most common manifestation of late LB in Europe (Aguero-Rosenfeld et al., 2005). These differences are likely due to the different species causing LB in the 2 continents (Aguero-Rosenfeld et al., 2005).

In the United States, where only B. burgdorferi sensu stricto is spread, a 2-step process is recommended by the Centre for Disease Control (CDC) (www.cdc.gov) when testing blood for evidence of Lyme disease in symptomatic patients. The first step uses a highly sensitive enzyme immunoassay (EIA) or immunofluorescence assay (IFA) followed, in the second step, by a standardized specific Western blotting assay when specimens result positive or equivocal by EIA or IFA. These tests may be falsely negative in patients with early disease and are not generally recommended when a patient presents with EM, but they are quite reliable for diagnosing later stages of disease (www.cdc.gov). In Italy, where the complex B. burgdorferi sensu lato is present, the available serologic methods are not sufficiently standardized, giving frequently false-positive and false-negative results, the latter especially in the local early stage of the disease. Accordingly to the Italian Ministry of Health guidelines, the use of the serologic assays as screening tests must be avoided and the clinical diagnosis may be formulated on the basis of anamnestic and epidemiologic data confirmed by direct laboratory assays to detect B. burgdorferi or its DNA (Ministero della Salute Italiano, 2000).

Among direct diagnostic assays, to reduce misdiagnosis, the utilization of molecular techniques has focused mainly on polymerase chain reaction (PCR)-based methods. After the first PCR assay reported in 1989 (Rosa and Schwan, 1989), various other PCR protocols were subsequently developed for the detection of B. burgdorferi sensu lato DNA in clinical specimens (Aguero-Rosenfeld et al., 2005, Ivacic et al., 2007, Joss et al., 2008, Portnoï et al., 2006, Postic et al., 2000).

The presence of LB has been described in various regions of Italy, especially Trentin, Veneto, and Central-Southern areas of the Italian peninsula (Ciceroni et al., 2001, Pugliese et al., 2007) and several cases of LB have been reported to the health authorities in Italy since 1990, when systematic national surveillance started (Tomao et al., 2005). Despite notification of LB in Italy is mandatory, the available information on the epidemiology and ecology of this disease derives from regional data and is still scanty, owing to inadequate notification, and little is known about occupational, recreational, and other behavioral risk factors (Tomao et al., 2005).

Recent epidemiologic data concerning the area of Italy where Parma is located (Northern Italy) are not available: this area was reported as non-endemic on 1997 having a seroprevalence of 0.2% (Santino et al., 1997). The aim of this study was to evaluate the presence of anti-B. burgdorferi antibodies and the presence of B. burgdorferi by cultivation and DNA detection in samples of subjects with clinical suspicion of LB attending the University Hospital of Parma (Italy) in the period 2002–2008.

Section snippets

Patients

A total of 2749 samples belonging to 2336 patients with the clinical suspicion of LB were analyzed at the Section of Microbiology of the Department of Pathology and Laboratory Medicine of the University Hospital of Parma, Italy, during the period 2002–2008.

In 2165 cases the symptoms and signs were generic (i.e., fever, headache, muscle or joint diffused pain). In an additional 171 cases, a specific suspicion of LB was supported by typical signs and symptoms of the disease (such as EM, knee

Results

Anti-B. burgdorferi antibodies (IgM and/or IgG) were detected by IFA in serum samples belonging to 277 patients and in CSF samples (only IgG) belonging to 3 patients, while EIA gave a positive result in serum samples belonging to 18 patients. In 24 cases, EIA result was equivocal for IgG only, IgM only, or both (Table 1). WB assay did not confirm the presence of anti-B. burgdorferi antibodies in 238 cases among the 319 positive or equivocal by EIA or IFA (Table 2). Specific anti-B. burgdorferi

Discussion

The general clinical picture of LB is similar in the United States and Europe. However, at least in part due to the organotropism of the Borrelia species, there are some differences concerning the frequency and the appearance of leading symptoms (Aguero-Rosenfeld et al., 2005, Franz and Krause, 2003, Tomao et al., 2005). In Italy where LB is a notifiable disease, the diagnosis based on clinical symptoms and epidemiologic data must be confirmed by direct laboratory assays (cultivation and/or DNA

Acknowledgments

This study was supported by the Ministry of University and Scientific Research Grant FIL, Parma, Italy.

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