Diagnosis of lyme borreliosis by polymerase chain reaction

Diagnosis of Lyme Borreliosis by Polymerase Chain Reaction MIRNA SˇITUM, MD, PhD GORAZD POJE, MD BLAZˇENKA GRAHOVAC, PhD ´ , MD, MS BRANKA MARINOVIC SONJA LEVANAT, PhD

B

orrelia burgdorferi (B. burgdorferi), a spirochete mostly transmitted by ticks of the Ixodidae family, causes Lyme borreliosis (LB), a multisystemic disease that affects the skin, joints, central nervous system, and heart.1,2 Typical and the most common skin manifestation of Lyme borreliosis is erythema migrans (EM), whereas neurologic symptoms, joint involvement, and chronic skin alterations can develop at later stages of the disease.3 In northwestern Croatia, an endemic area for Lyme borreliosis, four genomic B. burgdorferi sensu lato groups were identified in the Ixodes ricinus ticks: B. afzelii, B. garinii, B. valaisiana (group VS116), and B. burgdorferi sensu stricto.4 The classification of B. burgdorferi sensu lato into genomic groups has clinical relevance for LB. The association of B. afzelii with skin manifestations, of B. garinii with neurologic symptoms, and of B. burgdorferi sensu stricto with arthritis has been demonstrated.3,4 The pathogenetic potential of B. valaisiana has not yet been identified.5 In Croatia, B. burgdorferi was first isolated in 1991 at the Department of Dermatology and Venereology, Zagreb University Hospital Center, from the skin of an EM patient, and was named P1 Zagreb. Electrophoretic analysis of B. burgdorferi proteins revealed six of the most important proteins of different molecular mass: OspA, OspB, OspC, p41, p60, and p100, classifying the isolate into the B. burgdorferi sensu lato group.6 Sequence data and phylogenetic analysis confirmed that DNA isolates from the sera of patients with EM coming from northwestern Croatia belonged to the B. afzelii genospecies.7 The diagnosis of LB is generally based on clinical From the Department of Dermatology and Venereology, Sestre Milosrdnice University Hospital; the Department of Ear, Nose, and Throat, Zagreb University Hospital Center; the Croatian Institute of Transfusion Medicine; the Department of Dermatology and Venereology, Zagreb University Hospital Center; and the Department of Molecular Medicine, Ruder Bosˇkovic´ Institute, Zagreb, Croatia. Address correspondence to Mirna Sˇitum, MD, PhD, Zagreb University Hospital Center, Department of Dermatology and Venereology, Vinogradska 29, HR-10000, Zagreb, Croatia. E-mail address: [email protected] © 2002 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

picture and demonstration of specific antibodies to B. burgdorferi by indirect serologic tests. The immunofluorescence assay (IFA) measuring circulating serum antibody binding to B. burgdorferi antigen is most commonly used. Specific IgM antibodies to B. burgdorferi occur 2 to 4 weeks after infection (in most patients, initial IgM response decreases after 1–2 months), whereas the presence of specific IgG antibodies in serum can be demonstrated only at 6 to 8 weeks of infection, from whence they are detectable for months and years thereafter.3,8 The shortcomings of this assay are subjective interpretation of results, false-positive reaction in patients with treponemal infection and some autoimmune disorders,8,9 and low sensitivity in stage I disease.10 Low number of B. burgdorferi in pathologic lesions and tissue fluids, low antigen level, and the ability of B. burgdorferi to escape the host’s immune response result in slow, poor, and unreliable formation of specific antibodies.11 The interpretation of serologic test results in endemic populations with subclinical B. burgdorferi infection may frequently prove quite difficult.10,11 The method of target-sequence DNA amplification by repetitive cycles of DNA synthesis, polymerase chain reaction (PCR), has proved useful in the diagnosis of LB.12–14 It has proved especially valuable in the diagnosis of early dissemination of B. burgdorferi, spirochetemia, and from primary skin lesions, which is rarely manifested by clinical signs of advanced LB. In these cases, LB poses a diagnostic problem just because of the low B. burgdorferi concentration in blood, which makes isolation of B. burgdorferi unsuccessful, whereas PCR has proved to be fast, reliable, sensitive, and species-specific even in samples containing ⬍10 bacteria per milliliter of tested fluid.7,12–14 PCR has also been found useful when the clinical manifestations of LB are not typical, eg, absence of erythema chronica migrans (ECM), presence of isolated asymmetric joint lesions, or neuroborreliosis in the early stage of disease.15,16 Early results provided by PCR allow for and help in timely initiation of LB treatment as well as in the follow-up of therapeutic success.15 In addition, PCR has been used in 0738-081X/02/$–see front matter PII S0738-081X(01)00242-5

148 Sˇ ITUM ET AL.

typing of as-yet-unclassified B. burgdorferi genotypes17–19 as well as in assessing the possible persistence of B. burgdorferi infection activity. Therefore, the method is highly valuable in patients with chronic symptoms that are not specific exclusively for LB, such as arthritis and encephalomyelitis.16 –19 The aim of the present study was to analyze the sensitivity of IFA and PCR methods in the diagnosis of LB. Sensitivity testing was performed in three target groups of subjects at a high risk of LB infection (two groups of the risk population of forestry workers) or with existent clinical picture of LB. A group of subjects from the risk population of forestry workers from an LB-endemic area in northwestern Croatia, a group of subjects from the risk population of forestry workers from an LB-nonendemic area in Istria, and a group of subjects suffering from clinically manifest early LB in the form of ECM were included in the study. The fact that the inflammatory stage of circumscribed scleroderma (CS) and subsequent sclerotization resemble a typical late manifestation of LB, acrodermatitis chronica atrophicans, has stimulated some researchers to analyze sera of CS patients to identify the possible etiopathogenetic role of B. burgdorferi sensu lato in its development.20,21 Urged by this concept, we included CS patient serum testing in this study to demonstrate the presence of antibodies or Borrelia DNA.

Materials and Methods Three target groups of subjects were analyzed. The selection criteria were high risk of infection (two groups of the risk population of forestry workers) and overt or suspected clinical picture of LB. So the study included 82 subjects from the risk population of forestry workers from northwestern Croatia (Koprivnica and Bjelovar), an LB-endemic area, and 20 subjects from the risk population of forestry workers from an LB-nonendemic area in Istria and Opatija. The third group included patients with cutaneous manifestations of LB, ie, 40 patients with ECM, one patient with lymphadenosis cutis benigna (LCB), and five patients with CS. So 148 subjects were included in total. The study was carried out at the Department of Dermatology and Venereology, Zagreb University Hospital Center, and the Croatian Institute of Transfusion Medicine in Zagreb. A questionnaire designed for the study contained clinical and epidemiologic data (number and localization of tick bites), case history (time elapsed from tick bite to development of the clinical picture of disease, description of skin lesions upon tick bite and during the course of disease, time elapsed from tick bite to serum sampling, therapy), and dermatologic status (clinical picture of skin lesions at the time of examination). IFA testing of serum samples with B. afzelii antigen

Clinics in Dermatology

Y

2002;20:147–155

was performed at the Laboratory of Clinical Immunology, Immunofluorescence and Serodiagnosis, Department of Dermatology and Venereology, Zagreb University Hospital Center and Zagreb University School of Medicine in Zagreb. Suspension of B. afzelii, previously isolated from the skin of patients with ECM, was used for IFA. Serum samples were analyzed by PCR methods at the Croatian Institute of Transfusion Medicine.

Indirect Fluorescence Assay Lyophilized filtrate of Treponema reiteri culture (sorbent) was dissolved in buffered solution of NaCl, pH 7.4 (2 mg/ml buffer) and used for patient serum dilution (1:16). Then it was incubated for 30 minutes at 37°C and centrifuged for 5 minutes at 3000g in an attempt to isolate specific antibodies from the serum. Then serum dilution on microtiter plates was as follows: 1:32, 1:64, 1:128, 1:256, and 1:512. Buffered solution of NaCl, pH 7.4, was used for serum dilution. Then 10 ␮L of each serum dilution was applied to the slide with previously prepared antigen and incubated in a moist chamber for 30 minutes at 37°C, washed twice for 5 minutes in buffer, and left to dry. In the meantime, conjugates, ie, fluorescein-labeled anti-human IgM and fluoresceinlabeled antihuman IgG, were prepared. The conjugates were used at a 1:200 dilution (they were diluted in buffered solution of NaCl, pH 7.2). Ten microliters of conjugates was applied on each slide, reincubated for 30 minutes at 37°C in a moist chamber, washed in buffer three times for 5 minutes, and allowed to dry. Then reading of the results under a fluorescence microscope followed. A binocular Reichert dark-field microscope with E 2UG 1/1.5 exciting filter and GG 13/3⫹ 1-mm barrier filter was used. A CS 200 mercury lamp was used as a source of ultraviolet light. If the serum contained specific antibodies, the Borrelia fluoresced a yellow-green light; if there were no antibodies, the Borrelia did not fluoresce at all. The test was considered positive if the Borrelia fluoresced at a serum dilution ⱖ1:64 (IgM or IgG titer ⱖ64). Sera with a high titer of IgM and IgG antibodies (positive sera) and sera without IgM and IgG Borrelia antibodies (negative sera) were used as controls. The sera with positive IgG antibody titer on IFA (IgG antibody titer ⬎512) and negative IgM antibody titer on IFA (false-negative IgM IFA) were absorbed with antiIgG absorbent (rheuma factor sorbent) by 1:13 serum dilution with rheuma factor sorbent, left overnight at 4°C, and centrifuged on the next day for 10 minutes at 8000g. In this way, excess IgG antibodies were removed from the serum. Then, such sera were processed by Treponema reiteri culture filtrate (dilution 1:16), and IgM IFA was repeated as described above.

Clinics in Dermatology

Y

2002;20:147–155

Polymerase Chain Reaction DNA Isolation From Serum One milliliter of serum was centrifuged for 10 minutes at 15,000 rpm at room temperature to concentrate the fraction with bacteria. The precipitate was washed with 1 mL of phosphate-buffered saline, pH 7.0, and centrifuged at 12,000 rpm at room temperature for 5 minutes. Two hundred microliters of buffer II (100 ␮L NaCl 5 mol/L; 150 ␮L 10% sodium dodecyl sulfate; 2750 ␮L TE buffer, pH 8.0) and 10 ␮L proteinase K (10 mg/mL) was added to the precipitate and incubated for 2 hours at 50°C and then for 10 minutes at 95°C. After being cooled, 400 ␮L of a mixture of phenol, chloroform, and isoamyl alcohol was added, stirred for 5 minutes, and then centrifuged for 10 minutes at 12,000 rpm at room temperature. The aqueous phase was separated and DNA was precipitated with a double volume of 100% ethanol at ⫺20°C overnight. DNA precipitate was washed with 70% ethanol, dried in a vacuum centrifuge for 5 minutes, dissolved in 30 ␮L water with 1-hour incubation at 37°C, and stored at 4°C. Primer Design Two segments of the Borrelia species genome, one encoding ribosomal 16S RNA and the other a chromosomal gene for flagellin protein, were chosen as targets of amplification. The segment of ribosomal 16S RNA gene is highly preserved and broad-specific for eubacteria, and the selected primers have been described in the literature.22,23 The primers for broad-specific 16S RNA gene amplified a region of 215 base pairs (BS1 5⬘-GAGGAAGGTGGGGGATGA-3⬘; BS2 5⬘-GCCCGG GAACGGATTCAC). This amplification was used to control the yield and quality of DNA isolation.22 The primers for flagellin gene were created on the basis of multiple comparison of the sequences obtained from GenomeNet-DNA Database of the National Institute of Genetics, Kyoto, Japan, using BLAST software for optimal comparison of two sequences and the ClustalW software for multiple sequence comparison,23 which also provides output data for phylogenetic analysis based on sequence variation BAF 51 5⬘-GCTGCT GGCATGGGGGTTTCT-3⬘ (128 –149); BAF 31 5⬘-CAAT AGCATACTCAGTACTATTCT-3⬘ (832– 856); BAF 52 5⬘-CAGACAACAGAAGGGAATTTAAATG-3⬘ (219 – 244); BAF 32 5⬘-CAAGTGATGTATTAGCATCAA CTG-3⬘ (729 –754). To increase the method’s sensitivity, nested PCR, requiring synthesis of two primer pairs (external and internal), was chosen. The size of amplification product was 728 base pairs for the first amplification and 535 base pairs for the second amplification. DNA Amplification The final PCR components included 50 mmol/L KCl; 10 mmol/L TRIS (hydroxymethyl) aminomethane pH 8.3;

DIAGNOSIS OF LYME BORRELIOSIS

149

1.5 mmol/L MgCl2; 0.1% (wt/vol) gelatin; 100 ␮mol/L each of dATP, dGTP, dCTP, and dTTP; 2.5 U Amplitaq DNA polymerase (Perkin-Elmer, Branchburg, USA), and 20 pmol of each primer. The following conditions were used for amplification of ribosomal 16S RNA gene: initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 42°C for 30 seconds, and extension at 72°C for 30 seconds for 35 cycles, and final extension at 72°C for 7 minutes on a GeneAmp 2400 thermal cycler (Perkin-Elmer, Branchburg, USA). The conditions for flagellin gene amplification were as follows: initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 30 seconds for 30 cycles, and final extension at 72°C for 7 minutes. Because the flagellin gene was submitted to double amplification, 5 ␮L of the first PCR products were used in another round of amplification with new internal primers under the same amplification conditions. To test the specificity of PCR primers used in this study, DNA isolates from the following bacterial strains were tested: B. burgdorferi sensu lato and B. afzelii, Staphylococcus aureus, Enterococcus sp., S. epidermidis, Sarcina lutea, Klebsiella oxytoca, Escherichia coli, Proteus mirabilis, Yersinia sp., Salmonella enteritidis, Shigella sonnei, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus cereus, Treponema pallidum, and Leptospira interrogans. Single-Stranded Conformation Polymorphism Analysis Single-stranded conformation polymorphism analysis24 was performed using a 10% MiniClean gel system (Pharmacia, Uppsala, Sweden) under the conditions recommended by the manufacturer. Specific PCR product (4 – 6 ␮L) was mixed with 2 volumes of formamidedye solution (88% formamide, 10 mmol/L EDTA, 0.01% xylene cyanol), heat-denatured at 95°C for 5 minutes, and cooled rapidly in ice water (0°C, 5 minutes). Eight microliters of the denatured DNA solution was loaded onto a 10% polyacrylamide MiniClean gel. The electrophoresis conditions were 10 minutes at 200 V, 10 mA, and 5 W, and 2 hours at 375 V, 15 mA, and 10 W at 15°C. The gels were stained with silver.24,25 Sequencing PCR products of the flagellin gene (subcloned with Sure-Clone ligation kit, Pharmacia Biotech, Freiburg, Germany) were sequenced by the dideoxy chain-termination method25 using an AutoRead sequencing kit (Pharmacia Biotech) and fluorescent universal and reverse primers annealing to the multiple cloning sites of the pUC18 vector. Sequencing was carried out on an ALF DNA sequencer (Pharmacia Biotech, Freiburg, Germany).

150 Sˇ ITUM ET AL.

Table 1.

Clinics in Dermatology

Y

2002;20:147–155

Clinical Picture of Lyme Borreliosis According to Time Elapsed From Tick Bite to Serum Sampling

Time Elapsed From Tick Bite to Serum Sampling 1 day–6 weeks 6 weeks–4 months 4 months–several years No tick bite history Total

ECM

No. of Subjects

Endemic

Affected

LCB Affected

CS Affected

77 25 28 18 148

5 ... ... ... 5

23 16 1 ... 40

1 ... ... ... 1

... ... 3 2 5

ECM ⫽ erythema chronicum migrans; LCB ⫽ lymphadenosis cutis benigna; CS ⫽ circumscribed scleroderma.

Statistics Statistical analysis was done on an IBM computer with professional Statistica and Microstat Microsoft software. Methods of relative ratios and comparisons, ie, indexes and standard errors of proportions, were mostly employed because of the material specificity. Because the sample had attributive characteristics, the qualitative and quantitative results could only be processed by nonparametric statistical methods. The ␹2 tests for dependent and for independent samples were used.

Results Results Obtained by Clinical Methods Of 148 study subjects, 130 (87.84%) reported a tick bite, whereas 51 (34.45%) subjects had a clinical picture of LB in their dermatologic status. Development of the clinical picture of LB according to time elapsed from tick bite to serum sampling is presented in Table 1. Of 20 risk group subjects from the LB-nonendemic area, nine (45.00%) reported a tick bite, and none had a clinical picture of LB. Of 82 risk group subjects from the LBendemic area, 78 (95.12%) reported very frequent, multiple tick bites, while only five (6.09%) had a clinical picture of LB, manifested as ECM. In the group of 46 LB patients, ECM was present in 40 (86.96%), LCB in one (2.17%), and CS in five (10.87%) patients. Two (4.35%) subjects denied any history of tick bite.

Results Obtained by Laboratory Methods IFA Results These data refer to IFA positivity in study subjects who reported a tick bite but did not have a clinical picture of LB and to IFA positivity in LB patients according to data on tick bites. (Data on time elapsed between tick bite and serum sampling were of utmost relevance for the overall evaluation of IFA positivity and onset of the clinical picture.) Of 148 study subjects, positive IFA results were recorded in 61 (41.22%) subjects, five (8.33%) of them denying any tick bite. Clinical picture of LB was present in 31 subjects with positive IFA. IFA positivity according to time elapsed from tick bite to serum sampling is shown in Table 2.

In the group of subjects from the LB-nonendemic area, positive IFA was recorded in three (15%) subjects, all with IgG antibodies, while one subject did not report a history of tick bite. None of these subjects had the clinical picture of LB. In subjects from the LB-endemic area, 30 (37.80%) subjects had positive IFA, all with IgG antibodies, and two with concurrence of IgM antibodies. Two of these subjects denied any history of tick bite. Of the five subjects with ECM, three had positive IFA, indicating the presence of IgG antibodies. In the group of LB patients, IFA was positive in 28 (60.87%), pointing to IgG antibodies in 26, IgM antibodies in two, and both IgM and IgG antibodies in seven subjects. Twenty-two (47.83%) patients affected with ECM had IgG antibodies, while two had both IgG and IgM antibodies. The patient suffering from LCB (2.17%) also had positive IFA, with the presence of both IgM and IgG antibodies. All five (10.87%) CS patients had only IgG antibodies (Table 2). PCR Results PCR positivity is presented according to the history of tick bite, onset of clinical picture, and time elapsed from tick bite to serum sampling. Of 148 study subjects, 24 (16.22%) had positive PCR, all of them with a history of tick bite. Eleven of these 24 subjects with positive PCR also had a clinical picture of LB, and all had ECM. PCR positivity according to time elapsed from tick bite to serum sampling is shown in Table 3. None of the subjects from the LB-nonendemic area had positive PCR. Of 82 subjects from the LB-endemic area, 14 (17.07%) had positive PCR, only one of them Table 2. IFA Positivity According to Time Elapsed From Tick Bite to Serum Sampling Time Elapsed From Tick Bite to Serum Sampling 1 day–6 weeks 6 weeks–4 months 4 months–several years No tick bite history Total

Antibodies

No. of Subjects

IgM

IgG

IgM ⫹ IgG*

77 25 28 18 148

2 ... ... ... 2

23 20 9 7 59

3* 4* ... ... 7

IFA ⫽ immunofluorescence assay. *Patients also included in the group with IgG antibodies.

Clinics in Dermatology

Table 3.

Y

2002;20:147–155

DIAGNOSIS OF LYME BORRELIOSIS

151

Simultaneous PCR and IFA Positivity According to Time Elapsed From Tick Bite to Serum Sampling

Time Elapsed From Tick Bite to Serum Sampling 1 day–6 weeks 6 weeks–4 months 4 months–several years No tick bite history Total

IFA Antibodies No. of Subjects

PCR

IgM

IgG

IgM ⫹ IgG

77 25 28 18 148

13 7 4 ... 24

... ... ... ... ...

5 7 1 ... 13

... 1* ... ... 1

PCR ⫽ polymerase chain reaction; IFA ⫽ immunofluorescence assay. *Patient also included in the group with IgG antibodies.

with ECM in his medical history. In the group of 46 LB patients, PCR was positive in ten (21.74%) patients, all of them with a clinical picture of ECM. Compatibility of IFA and PCR IFA was positive in 13 of 24 subjects with positive PCR. Simultaneous positivity of PCR and IFA according to time elapsed from tick bite to serum sampling is presented in Table 3. In the group of subjects from the LB-endemic area, IFA was positive in six of 14 subjects with positive PCR, with IgG antibodies detected in all and IgM in none of them. Only one of 14 subjects with positive PCR had a clinical picture of LB, manifested as ECM. IFA was negative in this subject. IFA was positive in seven (70.00%) LB patients with positive PCR, with IgG antibodies detected in all, and both IgG and IgM antibodies in one of them (Table 3). Comparison of IFA and PCR Sensitivity Statistical analysis yielded a significant difference between IFA and PCR sensitivity within the group of subjects with positive history of tick bite (␹2 ⫽ 10.76; P ⬍ 0.005). In these subjects, IFA showed statistically significantly higher positivity than PCR, which was attributed to the detection of IgG antibodies also in the LB-nonendemic area (Fig 1). When the groups of subjects from LB-endemic area and of LB patients (ie, the only groups with positive PCR) were analyzed by ␹2 for dependent samples, the

Figure 1. Correlation of immunofluorescence assay and polymerase chain reaction in selected subjects with positive history of tick bite (n ⫽ 130)

between-group difference was not statistically significant (␹2 ⫽ 2.19; NS), indicating similar positivity of the two methods. This appears quite reasonable, because the groups included the high-risk population of forestry workers from the highly endemic area for LB characterized by a very high rate of B. afzelii infection and the patients with overt ECM picture. Therefore, positivity of both methods could have theoretically been expected in these specific groups of subjects. Sensitivity of the PCR method was observed to decrease, ie, to become statistically nonsignificant (␹2 ⫽ 1.49; NS) when the subjects from all risk groups (from both LB-endemic and -nonendemic areas) were compared with LB patients. Testing of the overall results obtained by IFA in the risk population (from both LB-endemic and -nonendemic areas) and in LB patients revealed IFA to be statistically significantly more sensitive (␹2 ⫽ 3.84; P ⬍ 0.05) than PCR, because IFA yielded higher positivity in all subjects recruited from the population at risk.

Discussion The high percentage of positive history of tick bite (87.8%) logically resulted from the specific selection of study subjects from the risk population of forestry workers and those suffering from cutaneous LB manifestations. Forestry workers are by far more than other occupations exposed to tick bites, and the spirochete B. afzelii is transmitted by tick bite to the skin of LB patients. In the risk population from the LB-nonendemic area, 45% of study subjects had a history of tick bite but none of them had a clinical picture of LB. The lower percentage of tick bite in the LB-nonendemic area (Istria, Opatija) could have been expected, although the subjects from this group were also recruited from the high-risk population of forestry workers, because the conditions in this geographic area (altitude, climate, and vegetation) are not favorable for ticks.26 The asymptomatic seropositivity in three of these subjects was not considered significant, the more so as none of them had positive PCR. The IgG antibodies detected probably resulted from B. afzelii infectivity, which had no significant pathogenetic potential for the disease to

152 Sˇ ITUM ET AL.

develop in the risk population from LB-nonendemic area, but its presence stimulated the formation of specific antibodies in the subjects.27,28 Positive history of tick bite was very common (⬎95%) in northwestern Croatia, an area of central Europe that is endemic for LB.4,29 Nevertheless, considering the high frequency of tick bites, the clinical picture of LB was not so common as could be expected. The clinical picture of LB was present in only five subjects from this group, supporting some reports stating that the infectivity of B. burgdorferi via tick bite is not so high for humans, eg, that ECM will develop in only one of 41 subjects sustaining an infected tick bite.28 All the five subjects from this group had ECM and reported a tick bite within the past 6 weeks, which seems logical because ECM is an early manifestation of LB. The clinical picture of LB in our subjects was consistent with those reported by other authors from neighboring countries and central Europe.28,29 Thus, ECM, the pathognomonic clinical form of LB, was also most commonly diagnosed in our study, ie, in 88.24% of study subjects. This percentage exceeds those reported by authors from central European countries, where it is around 56%, however analyzing the prevalence of ECM according to all manifestations of LB.4,28,29 In the present study, patients with exclusively cutaneous manifestations of LB were included and showed a very high prevalence of ECM. In all subjects, ECM occurred within 4 months from tick bite. Accordingly, it can be stated that in these patients ECM occurred at the stage of localized infection (early disease manifesting within 6 weeks from infected tick bite) or at the stage of disease dissemination (late disease manifesting in 4 months), when its occurrence is not infrequent either.4,28,30 LCB is a very rare skin manifestation of LB, which was only sporadically present in the total number of study patients. In the only patient with LCB, the onset of the disease occurred 1 month from the infected tick bite. Weber31 classifies this clinical form into stage II LB, although it may occur very early after infected tick bite. Namely, no consensus has yet been achieved on the time periods referring to particular stages of the disease.3 The very suspected role of B. burgdorferi in the etiopathogenesis of CS has stimulated us to investigate the possibility that morphea might in fact be a late form of LB.32–34 That is why CS patients were included in the study. Three of the five CS patients reported tick bite in the 1 to 3 years preceding the onset of the clinical picture of disease. It is clear because CS probably belongs to the late manifestations of LB, precisely, to the stage of persisting infection.33 Two patients could not recollect any tick bite, which is quite possible, because if bitten by a tick in the developmental stage of nymph, its presence on the skin is extremely difficult to perceive because of its small dimension and brief feeding.34 Besides this, the incubation is shorter for ECM and it is

Clinics in Dermatology

Y

2002;20:147–155

easier for the patient to remember a contact with a tick, whereas in stage II and III LB it may be difficult to relate current disease to a possible contact with a tick months or years before.32–35 Specific antibodies to B. afzelii detected by IFA were found in nearly 42% of the study population. This high rate of seropositivity is consistent with the data obtained in a study conducted in a part of northern Croatia, which yielded a 42.9% seropositivity in a risk group of forestry workers.36 Studies from Germany and Great Britain report on the rate of seropositivity in the same population of 17% and 25%, respectively.36,37 Our subjects from the LB-endemic area with positive IFA results had IgG antibodies, while two of them had both IgG and IgM antibodies. Only five subjects from this group had ECM, however, two of them with negative IFA results. Seronegativity in these patients was attributed to too short a time from infected tick bite (2–3 weeks) for the specific IgM antibodies to B. burgdorferi to develop. Similar results have been reported by Rusell et al,38 Shrestha et al,39 and Steere,28 who obtained negative IFA results in serum samples collected up to 3 weeks from the onset of ECM. Defense from B. burgdorferi infection in the early stage of disease is considered to proceed at the level of cutaneous immune reaction,40 thus specific antibodies being impossible to demonstrate in sera of these patients. According to Moffat et al,41 there is an enhanced activity of suppressor cells, and the occurrence of antibodies is restricted to the B. burgdorferi most immunogenic polypeptides. Obviously, the proportion of seropositive subjects in the endemic group by far exceeded the number of those with overt clinical picture.42 Seroepidemiologic studies of endemic populations have confirmed that the disease may have a subclinical course; ie, seropositivity for B. burgdorferi in high-risk groups has been clearly shown to considerably exceed that in the general population.43,44 Our results are consistent with these data. Analysis of the group of 45 affected subjects yielded 60.87% of IFA-positive subjects, 26 of them with IgG antibodies, two with IgM antibodies, and seven with both IgG and IgM antibodies. All subjects positive for IgM antibodies reported a tick bite in the preceding 6 weeks, and all had the clinical picture of early disease (ECM and LCB), as expected. The simultaneous occurrence of both IgM and IgG antibodies resulted from the development of new antibodies against different B. burgdorferi antigens.45,46 The predominant occurrence of IgG antibodies, ie, in all five CS patients and in others with ECM, was explained by the fact that immune response to IgM antibodies decreases in short term, whereas IgG antibodies are retained in the circulation for a long time, occasionally for months or years.47–50 According to some authors, antibodies to B. burgdorferi are found in 20% to 50% of CS patients,32,34,51 whereas others failed to demonstrate specific antibodies to B.

Clinics in Dermatology

Y

2002;20:147–155

burgdorferi in patients with CS.34,52,53 In our study, specific antibodies to B. burgdorferi were detected in all (100%) subjects. Controversial results reported from different studies put the role of B. burgdorferi in the etiology of CS under question, however, at the same time stimulating the research into its potential role in the etiopathogenesis of CS.53 The results of serologic testing in this group of subjects showed seropositivity in the early stage of disease (ECM) to be 78.57%, whereas literature data range between 10% and 50% of seropositive subjects.54 –56 It is so because most literature data refer to serodiagnosis based on commercial enzyme-linked immunosorbent assay, which is less sensitive than IFA that uses original lyophilized filtrate of B. burgdorferi culture grown in laboratory conditions.56 All our subjects with positive PCR results had a tick bite in their histories, whereas only 11 had a clinical picture of LB and all showed ECM. The PCR positive results according to time elapsed from tick bite are of highest importance, because the method is known to be very useful in demonstrating both early and late persisting B. burgdorferi infection.57 Study results revealed 54.17% of study subjects to have sustained a tick bite up to 6 weeks before (early infection), and the rest of 45.83% subjects about a year before (persisting infection), which is consistent with literature data.57 In the group of risk subjects from the LB-endemic area, 17% had positive PCR results, ie, only one subject with positive PCR result had ECM. The presence of B. afzelii was determined in the others; however, the clinical picture of LB was absent. This might speak in favor of the early detection of B. afzelii infection and possibility of timely treatment. In the group of LB patients, there were ten (21.74%) subjects with positive PCR results. They all had ECM that had occurred 2 to 4 months from tick bite, so the diagnosis was simply confirmed by PCR. The compatibility and sensitivity of IFA and PCR appear to deserve a few words more. In Croatia, the PCR method was introduced in the detection of B. afzelii in 1998, and our intention was to assess the method’s sensitivity and reliability relative to the standard serodiagnosis by use of IFA. Analysis of simultaneous IFA and PCR positivity revealed only 13 of 24 PCR-positive subjects to be positive on IFA, and they all had IgG antibodies. In the group of subjects from the LB-endemic area, six of 14 PCR-positive subjects had positive IFA, only one of them with ECM. These results could be considered as supporting the early detection of B. burgdorferi infection, thus rendering a timely introduction of treatment possible. Also, IFA was not positive in the patient with ECM, as it must have been the early-stage disease when specific antibodies to B. burgdorferi had not yet been produced or had not yet reached a detectable level

DIAGNOSIS OF LYME BORRELIOSIS

153

in the patient’s serum. Other subjects from this group with both PCR- and IFA-positive results reported a tick bite within up to 1 year before and had a high titer of IgG-specific antibodies to B. burgdorferi in their sera, which allowed them to be properly treated. This is what makes PCR a very valuable method indeed. In the group of LB patients, there were ten PCR-positive subjects, seven of them with positive IFA results and all with ECM. In these patients, the infection with B. burgdorferi was not dubious. The question is how to explain IFA positivity in all CS patients with simultaneously obtained negative PCR results. Do these results indicate that PCR is a tissuespecific method that does not produce reliable results on serum analysis? Did IFA positivity result from a coincidence, since all CS patients were diagnosed in an LB-endemic area where a high rate of B. afzelii infectivity is a usual finding? Did perhaps the finding of IgG antibodies result from their prolonged persistence in the circulation even after therapy administration; thus, a positive PCR result could not be expected at all, or is morphea a chronic, noninfective collagenosis? The literature offers controversial data on the association of B. burgdorferi and CS. Because CS in its involutional stage completely mimics a typical clinical manifestation of LB, ie, acrodermatitis chronica atrophicans, some authors have embarked on investigating the role of B. burgdorferi in its genesis. Only some succeeded to cultivate B. burgdorferi sensu lato in biopsy material from CS lesions, some have reported excellent results in the management of CS by the antibiotics used in therapy for LB, and some demonstrated the presence of specific antibodies to B. burgdorferi sensu lato and strong lymphoproliferative response to B. burgdorferi in CS patients. Others, however, failed to demonstrate any association between B. burgdorferi sensu lato infection and the occurrence of CS. The methods used to demonstrate this association certainly require further improvement and supporting techniques, and this task is now awaiting our efforts to try to solve it.54 –56 Testing of the pooled results obtained by IFA in the risk population (from both LB-endemic and -nonendemic areas) and in the LB patients showed IFA to have a statistically significantly higher sensitivity, because it yielded higher positivity in all subjects from the risk population. Statistical analysis pointed to a significant sensitivity difference between IFA and PCR also within the large group of subjects (n ⫽ 130) with a positive history of tick bite. In these subjects, IFA showed a significantly higher positivity than PCR. The reason for this was that the presence of IgG antibodies was frequently recorded also in the LB-nonendemic area, where it merely indicated infection with inadequately pathogenic B. afzelii rather than active disease, because the presence of active infection would have been demonstrated by PCR.

154

Sˇ ITUM ET AL.

Testing of the results obtained in the groups of subjects from the LB-endemic area and of LB patients (ie, groups in which PCR positivity exclusively occurs) by the test for dependent samples yielded no statistically significant difference between IFA and PCR. It appears reasonable, as these included only the high-risk population of forestry workers from an extremely endemic area for LB, where the rate of B. afzelii infectiousness is very high, and the group of patients with overt picture of ECM. Both methods are theoretically expected to produce positive results in either of these specific subject groups.

References 1. Burgdorfer W, Barbour AG, Hayes SF, et al. Lyme disease: a tick-borne spirochetosis? Science 1982;216:1317–9. 2. Burgdorfer W. Discovery of the Lyme disease spirochete and its relationship to tick vectors. Yale J Biol Med 1984; 57:71– 6, 515–20. 3. Sˇ itum M. Lyme bolest. In: Lipozenc˘ ic´ J, editor. Dermatovenerologija. Zagreb: Naklada Zadro, 1999:101–3. 4. Golubic´ D, Rijpkema S, Tkalec-Makovec N, Ruz˘ ic´ E. Epidemiologic, ecologic and clinical characteristics of Lyme borreliosis in Northwest Croatia. Acta Med Croat 1998;52: 7–13. 5. Wang G, Van Dam AP, Le Fleche AL, et al. Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19). Int J Syst Bacteriol 1997;47:926 –32. 6. Bolanc˘ a-Bumber S, Sˇ itum M, Balic´ -Winter A, et al. First isolation of Borrelia burgdorferi sensu lato in Croatia. Acta Dermatovenerol Croat 1997;5:95–9. 7. Sˇ itum M, Grahovac B, Markovic´ S, et al. Detection and genotyping of Borrelia burgdorferi sensu lato by polymerase chain reaction. Croat Med J 2000;41:47–53. 8. Magnarelli LA, Anderson JF, Johnson RC. Cross-reactivity in serological tests for Lyme disease and other spirochetal infections. J Infect Dis 1987;156:183– 8. 9. Berger BW. Laboratory tests for Lyme disease. Dermatol Clin 1994;12:19 –24. 10. Shrestha M, Grodzicki RL, Steere AC. Diagnosing early Lyme disease. Am J Med 1985;78:233– 40. 11. Berg D, Abson KG, Prose NS. The laboratory diagnosis of Lyme disease. Arch Dermatol 1991;127:866 –70. 12. Lebech AM, Hansen K, Brandrup F, Clemmensen O, Halkier-Sorensen L. Diagnostic value of PCR for detection of Borrelia burgdorferi DNA in clinical specimens from patients with erythema migrans and Lyme borreliosis. Mol Diagn 2000;5:139 –50. 13. Malloy DC, Nauman RK, Paxton H. Detection of Borrelia burgdorferi using the polymerase chain reaction. J Clin Microbiol 1990;28:1089 –93. 14. Saiki RK, Gelfland DH, Stoffer S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487–91. 15. Goodman JL, Bradley JF, Ross AE, et al. Bloodstream invasion in early Lyme disease: results from a prospective, controlled, blinded study using the polymerase chain reaction. Am J Med 1995;99:6 –12.

Clinics in Dermatology

Y

2002;20:147–155

16. Christen HJ, Eiffert H, Ohlenbusch A, Hanefeld F. Evaluation of the polymerase chain reaction for the detection of Borrelia burgdorferi in cerebrospinal fluid of children with acute peripheral facial palsy. Eur J Pediatr 1995;154: 374 –7. 17. Zbinden R, Goldenberger D, Lucchini GM, et al. Comparison of two methods for detecting intrathecal synthesis of Borrelia burgdorferi-specific antibodies and PCR for diagnosis of Lyme neuroborreliosis. J Clin Microbiol 1994;32: 1795– 8. 18. Muellegger RR, Zoechling N, Soyer HP, et al. No detection of Borrelia burgdorferi-specific DNA in erythema migrans lesion after minocycline treatment. Arch Dermatol 1995;131:678 – 82. 19. Liveris D, Gazumyan A, Schwartz I. Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis. J Clin Microbiol 1995;33: 589 –95. 20. Weide BA Weide B, Walz T, Garbe C. Is morphea caused by Borrelia burgdorferi? Br J Dermatol 2000; 142:636 – 44. 21. Ozkan S, Atabey N, Fetil E, Erkizan V, Gunes AT. Evidence for Borrelia burgdorferi in morphea and lichen sclerosus. Int J Dermatol 2000;39:278 – 83. 22. Wilske B, Preac-Mursˇ ic´ V, Jauris S, et al. Immunological and molecular polymorphism of OspC and immunodominant major outer surface protein of Borrelia burgdorferi. Infect Immun 1993;61:2182–91. 23. Assous MV, Postic D, Paul G, et al. Individualisation of two new genomic groups among America Borrelia burgdorferi sensu lato strains. FEBS Microbiol Lett 1994;121: 93– 8. 24. Orita Miwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphism. Proc Natl Acad Sci USA 1989;86:2766 –70. 25. Mohabeer AJ, Hiti AL, Martin WJ. Non-radioactive single strand conformation polymorphism (SSCP) using the Pharmacia ‘PhastSystem.’ Nucleic Acids Res 1991;19:3154. 26. Poljak I, Trosˇ elj-Vukic´ B, Miletic´ B, et al. Low seroprevalence of Lyme borreliosis in the mountainous area of Gorski Kotar, Croatia. Croat Med J 2000;41:433– 6. 27. Rijpkema SGT. Diagnosis and transmission of Lyme borreliosis. Dissertation. Utrecht: Universita¨ t Utrecht, 1995. 28. Steere AC. Lyme disease. N Engl J Med 1989;321:586 –96. 29. Strle F, Cimperman J, Pleterski-Rigler D, et al. Lyme borelioza u Sloveniji. In: Ropac D, editor. Lyme borelioza u Jugoslaviji. Zagreb: Zbornik MAH, 1989.93–100. 30. Buchner SA, Lautenschlager S, Itin P, et al. Lymphoproliferative responses to Borrelia burgdorferi in patients with erythema migrans, acrodermatitis chronica atrophicans, lymphadenosis benigna cutis, and morphea. Arch Dermatol 1995;131:673–7. 31. Weber K. Cutaneous manifestations of Lyme Borreliosis in Europe. J Spiroch Tick-Borne Dis 1994;1:57– 60. 32. Aberer E, Klade H, Stanek G, Gebhard W. Borrelia burgdorferi and different types of morphea. Dermatologica 1991;182:145–54. 33. Aberer E, Neumann R, Stanek G. Is localized scleroderma a Borrelia infection? Lancet 1985;2:278. 34. Buechner SA, Winkelmann RK, Lautenschlager S, et al.

Clinics in Dermatology

35.

36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

Y

2002;20:147–155

DIAGNOSIS OF LYME BORRELIOSIS

Localized scleroderma associated with Borrelia burgdorferi infection. J Am Acad Dermatol 1993;29:190 – 6. Fahrer H, Sauvain MJ, vd Linden S, et al. Prevalenz der Lyme-Borreliose in einer Schweizerischen Risiko-Population. Schweiz Med Wochenschr 1988;118:65–9. Burek V, Misˇ ic´ -Majerus LJ, Maretic´ T. Antibodies to Borrelia burgdorferi in various population groups in Croatia. Scand J Infect Dis 1992;24:683– 4. Hamlet N, Nathwani D, Ho-Yen DO. Borrelia burgdorferi infection in UK workers at risk of tick bites. Lancet 1989; 1:790. Russell H, Sampson JS, Schmid GP, et al. Enzyme linked immunosorbent assay and indirect immunofluorescent assay for Lyme disease. J Infect Dis 1984;149:465–70. Shrestha M, Grodzicki RL, Steere AC. Diagnosis of early Lyme disease. Am J Med 1985;78:235– 40. Buchner A, Rufli T. Erythema chronicum migrans: evidence for cellular immune reaction in the skin lesion. Dermatologica 1987;174:144 –9. Moffat CM, Sigal LH, Steere AC, et al. Cellular immune findings in Lyme disease: correlation with serum IgM and disease activity. Am J Med 1984;77:625–32. Fahrer H, Van der Linden SM, Sauvain MJ, et al. The prevalence and incidence of clinical and asymptomatic Lyme borreliosis in a population at risk. J Infect Dis 1991;163:305–10. Guy EC, Batemann DE, Martyn CN, et al. Lyme disease: prevalence and clinical importance of Borrelia burgdorferi specific IgG in forestry workers. Lancet 1989;1:484 – 6. Morgan-Capner P, Cutler SJ, Wright DJM. Borrelia burgdorferi infection in UK workers at risk of tick bites. Lancet 1989;1:789. Craft JE, Fisher DK, Shimamoto GT, Steere AC. Antigens of Borrelia burgdorferi recognized during Lyme disease: appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J Clin Invest 1986;78:934 –9.

155

46. Datwyiler RJ, Luft BJ. Immunodiagnosis of Lyme borreliosis. Rheum Dis North Am 1989;15:727–34. 47. Grodzicki RL, Steere AC. Comparison of immunoblotting and indirect enzyme-linked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease. J Infect Dis 1988;157:790 –7. 48. Dattwyler RJ, Volkman DJ, Luft BJ, et al. Seronegative Lyme disease: dissociation of specific T- on B-lymphocyte responses to Borrelia burgdorferi. N Engl J Med 1988;319: 1441– 6. 49. Barbour AG. Laboratory aspects of Lyme borreliosis. Clin Microbiol Rev 1988;1:399 – 414. 50. Wilske B, Schierz G, Preac-Mursic V, et al. Serological diagnosis of erythema chronicum migrans disease and related disorders. Infection 1984;12:331–7. 51. Rufli T, Lehner S, Aeschlimann A, et al. Zum Erweiterten Spektrum zeckenubertragener Spirocha¨ tosen. Hautarzt 1986;37:597– 602. 52. Hansen K, Serup J, Hoybye S. Antibodies to Borrelia burgdorferi and localized scleroderma. Lancet 1987;1: 682. 53. Fan W, Leonardi CL, Penneys NS. Absence of Borrelia burgdorferi in patients with localized scleroderma (morphea). J Am Acad Dermatol 1995;33:682–5. 54. Duffy J, Mertz LE, Wobig GH, Katzmann JA. Diagnosis of Lyme disease: the contribution of serologic testing. Mayo Clin Proc 1988;63:1116 –21. 55. Cieselski CA, Markowitz LE, Horsley R, et al. Lyme disease surveillance in the United States, 1983–1986. Rev Infect Dis 1989;11(Suppl 6):S1435– 41. 56. Hansen K, Asbrink E. Serodiagnosis of erythema migrans and acrodermatitis chronica atrophicans by Borrelia burgdorferi flagellum enzyme-linked immunosorbent assay. J Clin Microbiol 1989;27:545–51. 57. Kahl O, Gern L, Gray IS, et al. Detection of Borrelia burgdorferi sensu lato ticks: immunofluorescence assay versus polymerase chain reaction. Zentralbl Bacteriol 1998;287: 205–10.

N

ickel, the most common metal allergen, has been used in United States coinage for over one hundred years. The first nickel coin was the three cent piece, minted between 1865–1889. The composition of the coin was 75% copper and 25% nickel. The nick name copper already used for the English one cent piece, therefore the three cent coin was nick named the nickel. Due to post war inflation the Three cent coin lost popularity and was discontinued in 1889.1

From the collection of Raymond T. Kuwahara, MD, Memphis, TN. REFERENCE: 1. Mishler C. Coins, questions and answers. 4th ed. New York, Golden Books, 1998:64-5.