Introduction
Lyme borreliosis has been reported throughout Europe where it is the most
common tickborne infection, as it is in the United States [1].
Clinically, it shows up as a multisystemic disease, presenting dermatological,
rheumatic, neurological and cardiac manifestations.
The first reported human case of Lyme disease in Portugal was identified
in 1989 [2]. Diagnosis is preformed by the Centre for Vectors and Infectious
Diseases Research (CEVDI) at the Instituto Nacional de Saúde Dr.
Ricardo Jorge (National Institute of Health, INSA), using several techniques
including culture, PCR, and antibody detection. The first strains of Borrelia
burgdorferi sensu lato were isolated from ticks captured in the south
of Portugal [3] and the study showed that they belong to a new species, B.
lusitaniae [4]. Subsequent studies confirm the presence of several B.
burgdorferi s.l. species (B. lusitaniae, B. afzelii, B. garinii and B. valaisiana)
in ticks and the infection prevalence could vary between: studies have
found prevalences of 11.9% (n= 234, collected in several regions), 11.8%
(n= 2806, Mafra region), 34.7% (n=206, Grandola region); and 31.2% (n=285,
the island of Madeira) [5, 6, 7, 8]. In all the studies made so far, B.
lusitaniae is the most prevalent borrelia species. Recently, a strain of
this species was isolated from a human sample, indicating that it could
cause disease in humans [9]. Other species of borrelia, B. garinii,
B. afzelii B. burgdorferi sensu stricto and B. valaisiana have
already been detected in mainland Portugal and/or the island of Madeira
[5, 10]. Since 1999, Lyme borreliosis has been a mandatorily notifiable
disease in Portugal, but only a few cases are reported each year, which
does not allow consistent analysis of risk factors and the impact on public
health. The aim of this study was to contribute to a more precise evaluation
of the epidemiological situation of Lyme borreliosis in Portugal, analysing
the data available at the CEVDI’s laboratory concerning the serological
diagnosis of this disease and data available on the statutory notifiable
disease register.
Material and methods
The results of previous testing of all the sera and/or cerebrospinal
fluid (CSF) of patients with clinical suspicion of Lyme borreliosis
received at CEVDI’s laboratory between 1990 and 2004 were
analysed retrospectively. The antibodies were detected by indirect
immunofluorescence in-house assay using a strain of B. garinii
and a cut-off of 1:256 for IgG in sera and 1:4 in CSF were adopted.
All borderline and positive samples were confirmed by immunoblot
assay also an in-house test, using a strain of B.garinii. The
interpretation was done according to the European group recommendations
[11].
All the positive sera were tested to Treponema spp. and rheumatoid factor
and all sera with a positive result were considered to be false positives
for Lyme borreliosis. The laboratory definition of a positive case is
when we detected a seroconversion (significant change in levels of the
specific antibodies IgG and/or IgM in two samples), or when we detected
a positive titres of specific antibodies in one sample, in patiens with
clinical suspicion of Lyme borreliosis [12].
The data from the laboratory confirmed positive cases were compared with
the available data from the cases of Lyme borreliosis notified during
the period of 1999-2004. The notification of human cases of Lyme borreliosis
was done directly by the clinician to the competent health authority,
the Direcção Geral de Saúde (DGS), at the health
Ministry. The case definition establish to the clinicians by the health
authority must fit the following criteria. Confirmed case: Erythema migrans
confirmed by laboratory findings or at least one of the late manifestations
of Lyme borreliosis with laboratory confirmation
Results
Among 12 535 biological samples taken for analysis from patients with
clinical suspicion of Lyme borreliosis, 628 (5%) tested positive using
the EUCALB diagnostic criteria.
In patients with neurological symptoms, CSF was sometimes sent for analysis
(21%). Data is available describing the 628 Portuguese patients, 129
of whom tested positive for both CSF and sera. The remaining 499 patients
were diagnosed based in the result of sera analysis, with the observation
of seroconversion. The number of cases per year varied between 2 and
78, with the highest number of cases in 1997 [FIGURE 1].
The geographical distribution of the positive cases, based in
the patients’ home addresses, shows that Lyme borreliosis
infection has been seen in 17 of the 20 districts of Portugal
[FIGURE 2].
There were slightly more female patients (53.5%) than male patients
(46.5%).
The notification forms were frequently not filled in completely, which
may have caused some distortion in the data analysis of age and clinical
manifestations. Information on patient age was available on only 62.3%
of the forms. Analysing the available data, the mean age was 44 years
old (range: 2 months to85 years) and the age group most affected was
35-44 years old (21.3%) [FIGURE 3].
No clinical symptoms were reported in 237 (37.7%) of the 628
positive cases ([FIGURE 4]. Analysis of the information provided
by the physician in the remaining 391 cases showed that the most
frequently reported manifestations were neurological, reported
in 146 patients (37.3%), followed by nonspecific symptoms in
109 cases (27.8%). Five of the cases with nonspecific symptoms
had hepatic symptoms (4.5%), nine had myalgia (8.3%), 19 had
optical symptoms (17.4 %) and 76 reported only fever (69.7%).
The evaluation of the number of cases reported nationally between
1999 and 2004 (n=24) [13] and the number of positive cases confirmed
by our laboratory (n=225) during the same period, show that is
clearly an underreported disease. The annual incidence, estimated
on the basis of the statutory notifiable disease is 0.04 per
100 000 inhabitants. However, when laboratory data are taken
into account, we assume that this rate could be on average 10
times higher, 0.4 per 100 000 inhabitants [FIGURE 5].
Discussion/Conclusion
Although Lyme borreliosis is a mandatorily notifiable disease in Portugal,
the evaluation of CEVDI data concerning human cases of Lyme borreliosis
and the number of notified cases during the same period (1999-2004) shows
that this disease, like other vector borne diseases, such as boutonneuse
fever (the most prevalent tick borne disease in Portugal), is clearly
underreported in our country [14]. According to our data, between 1999-2004
we detected an average of 35 new cases of Lyme borreliosis each year.
Other diseases such as AIDS and tuberculosis have a bigger impact on
public health and the general impression gained is that Lyme borreliosis
cases are not considered important enough to notify and to publish.
The major problem of underreporting is the impossibility of realise an
epidemiological analysis of Lyme borreliosis in Portugal. For example,
according to the notification data, Lyme borreliosis is more common in
the Braga district (n=14) in northern Portugal, but when the results
are analysed, the only sample from this district to be sent for analysis
was negative and the districts showing higher number of confirmed cases
are Lisbon (n=286), Setubal (n=133) and Evora (n=65) districts located
in central and southern Portugal. It is also possible that the results
have been influenced by the proximity of the CEVDI’s facilities
to these regions, and the hospitals and physicians located at Northern
regions of Portugal may usually send their samples to other regional
laboratories that also perform these tests. For example, if sufficient
samples from Braga district and other northern regions were sent to our
laboratory, perhaps the proportion of positive cases in these regions
would increase. Also, if we analyse not only the number of positive cases
but also the proportion of it, the district of Lisbon is simultaneously
the district with a higher number of positive cases and one of the districts
with a lower proportion of positive cases.
As the laboratory data are not cross-checked with the official data,
it is impossible to know which cases detected at CEVDI were reported
to the health authorities, which laboratories performed the laboratory
testing and why the clinicians did not notify the positive cases that
they diagnose. Also, the fact that some of the positive cases may have
been in patients who acquired their infections in districts or countries
other than their area of residence should be considered, although patients
in Portugal usually use the health facilities in their area of residence.
In our experience, fewer than 10 patients during the time period considered
(1999-2004) mentioned the possibility that they may have acquired their
infection outside of their area of residence. However, the number of
positive cases of Lyme borreliosis detected is undoubtedly higher than
the number of cases reported. The reported incidence of Lyme borreliosis
in Portugal is among the lowest reported in Europe. However, if we analyse
the proportion of positive cases detected during this study (5%), we
can see that this value is similar to the detected in other studies of
seroprevalence in risk populations performed in several European countries
[15]. After 15 years performing laboratory diagnosis, even knowing the
limitations of laboratory results and being aware that the diagnosis
of Lyme borreliosis should be always established by the clinician, these
data, could contribute to the better understanding of the epidemiology
of Lyme borreliosis in our country. To improve the notification of this
disease, a network should be established to link all laboratories performing
Lyme borreliosis diagnosis, aggregating all laboratory detected cases.
This would allow the competent health authority to compare this information
with the cases notified by clinicians and to make a more accurate analysis.
The distribution of positive cases is influenced by clinicians’ awareness
of vector borne diseases, but the size of the I. ricinus population and
the prevalence of infected ticks are also contributory factors to the
incidence of the disease. The estimated annual incidence for Lyme borreliosis
in Portugal is 0.04 per 100 000 inhabitants. A higher estimated can be
obtained if we take laboratory data into consideration (0.4 per 100 000
inhabitants). However, as other laboratories also perform this test,
it seems likely that underreporting is even higher, and consequently
the true incidence of Lyme borreliosis in Portugal should be similar
to the published values detected in other countries such as Scotland
(0.6 per 100 000 inhabitants), United Kingdom (0. 3 per 100 000 inhabitants)
and much lower than that detected in countries such as France (16 per
100 000 inhabitants), Germany (17.8-25 per 100 000 inhabitants), Bulgaria
(55 per 100 000 inhabitants), Slovenia (120 per 100 000 inhabitants)
and Austria (130 per 100 000 inhabitants) [1, 16, 17]. It would be interesting
to compare the incidence detected in Portugal with geographical areas
such as southern Spain, Morocco, Tunisia and Algeria but, to our knowledge,
there are no available data published concerning the incidence of the
disease in the these regions. All these areas share with Portugal some
eco-epidemiological aspects such as vector population abundance and prevalence
of infection, lack of information about the vertebrate reservoirs and
the presence of the different Borrelia burgdorferi s.l. strains with
particular relevance to B. lusitaniae., During the past five years, the
number of human cases detected each year at CEVDI seems to have stabilised
at approximately thirty five cases per year. This reduction may perhaps
be explained by the increased number of other laboratories performing
this diagnosis. Also, due to the diversity of the possible clinical presentations
of Lyme borreliosis that may be confused with other aetiologies, the
benign course of the majority of clinical cases, and the usually very
positive response to the timely application of antibiotics, a large percentage
of cases are never sent to the laboratory to confirm a clinical diagnosis.
In this study, the positive cases which mention erythema migrans are
very rare, probably because many clinicians are aware that this stage
frequently does not evoke an antibody response and that laboratory confirmation
cannot be expected, and therefore do not request a laboratory confirmation
of their clinical diagnosis. Considering that the incidence of Lyme borreliosis
is directly linked to the density of the tick vector I. ricinus, and
knowing that this species is not found in high tick population densities,
we would expect the incidence of Lyme borreliosis to also be low. However,
we should also consider the I. ricinus has been found to exist all over
the country, but due to differing environmental characteristics, especially
climate, distribution is not uniform throughout Portugal but focused
in some regions where conditions are more suited to the survival of this
tick species, and where this species predominates, achieving high population
density.
In the absence of publications describing clinical cases, the information
available in the clinical forms is very useful because the analysis allows
us to clarify some epidemiological aspects such as risk factors concerning
age, sex and geographic localisation.
Other information that would help laboratory diagnosis, such as symptom
onset date, information about occurrence of recent tick bites, and recent
trips, are frequently unavailable. This is why collaboration and exchange
of information between clinicians and laboratories are so important.
Research concerning the eco-epidemiology of Lyme borreliosis in Portugal
has so far been slow to advance, and it is difficult to study the impact
and risk factors. However this knowledge is essential if we are to implement
adequate prevention programmes, which are currently considered the best
approach to solving the problem of vectorborne diseases. Sixteen years
after the report of the first human case of Lyme borreliosis in Portugal
this is still a poorly understood disease in Portugal. Due to the scattered
distribution of the positive human cases and the scattered nature of
the tick vector distribution throughout Portugal, the most effective
prevention measure for Lyme borreliosis in Portugal is probably educating
risk groups about how to avoid tick bites.
Acknowledgements
The authors wish to thank to Dr. Joseph Piesman, CDC, USA, and
Dr. Teresa Paixão, INSA, Portugal, for helpful editing
on this manuscript.
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