Borrelia burgdorferi Infection and Cutaneous Lyme Disease, Mexico

[ladycolorado]

Suggested citation for this article: Gordillo-Pérez G, Torres J, Solórzano-Santos F, de Martino S,
Lipsker D, Velázquez E, et al. Borrelia burgdorferi infection and cutaneous Lyme disease,
Mexico. Emerg Infect Dis. 2007 Oct; [Epub ahead of print]
Borrelia burgdorferi Infection and
Cutaneous Lyme Disease, Mexico
Guadalupe Gordillo-Pérez,* Javier Torres,* Fortino Solórzano-Santos,* Sylvie de Martino,†
Dan Lipsker,† Edmundo Velázquez,* Guillermo Ramon,* Muñoz Onofre,* and Benoit Jaulhac†
*Instituto Mexicano del Seguro Social, Mexico City, Mexico; and †Hôpitaux Universitaires, Strasbourg, France
Four patients who had received tick bites while visiting forests in Mexico had skin lesions that met
the case definition of erythema migrans, or borrelial lymphocytoma. Clinical diagnosis was supported
with histologic, serologic, and molecular tests. This study suggests the Borrelia burgdorferi infection
is in Mexico.
Lyme disease is the most frequently reported vectorborne infectious disease in the United
States and Europe (1,2). Studies have suggested that Borrelia burgdorferi infection might be
endemic to Mexico (3,4). We searched for histologic, immunologic, and molecular evidence of
B. burgdorferi infection in patients with cutaneous manifestations suggestive of Lyme disease in
Mexico.
The Study
From June 1999 to October 2000, 4 patients in Mexico City had clinical manifestations
suggestive of Lyme disease (5,6). Two (36 and 54 years of age) had erythema migrans lesions,
and 2 (9 and 34 years of age) had borrelial lymphocytoma lesions. Two reported having been
bitten by a hard tick; the other 2, by a nonflying insect. Bites occurred while camping in forests:
3 near Mexico City (National Park La Marquesa) and 1 in Quintana Roo, a southern state in
Mexico. All patients lived in Mexico City and had never traveled outside Mexico.
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Two patients were treated for acute skin lesions (consistent with erythema migrans),
malaise, and arthralgia. The skin lesion was an erythematous macula with regular, reddish edges
and a pink center. One patient had a 5-cm lesion on the left forearm; the other had a 6-cm lesion
on the left thigh. For the 2 other patients, a nodular erythematous cutaneous lesion (consistent
with lymphocytoma), 0.5–2 cm in diameter with regular edges, developed 2 months after the
bite. One patient’s lesion was on the earlobe; the other’s, on the left cheek.
Serum from each patient was tested for immunoglobulin M (IgM) and IgG against B.
burgdorferi sensu lato by using a commercial ELISA (cutoff optical density 0.200 and
indeterminate 0.200–0.400) (Enzygnost Borreliosis, Dade Behring, Marburg, Germany) (7). A
Treponema pallidum ELISA (Abbott Murex, Wiesbaden, Germany) was performed to rule out
cross-reaction with T. pallidum infection. Serum samples positive for B. burgdorferi by ELISA
were further tested by Western blot (WB) by using the Marxblot assay (MarDx Diagnostics,
Carlsbad, CA, USA) and Centers for Disease Control and Prevention (CDC) criteria (5).
Serum samples from the 2 lymphocytoma patients were positive for B. burgdorferi by
ELISA and WB (Figure 1, panel A; Table). For the 2 erythema migrans patients, serum samples
taken 2 weeks after the tick bite were negative for B. burgdorferi IgM and IgG; but 2 months
later, 1 patient became seropositive, confirmed by WB (Figure 1, panel B; Table).
Histologic examination of skin biopsy specimens from each erythema migrans lesion
showed a mononuclear cell infiltrate in the superficial and deep dermis; infiltrate included
lymphocytes and plasma cells around the perivascular zones. Biopsy samples of lymphocytoma
lesions showed dense nodular lymphocytic infiltrates in the reticular dermis with well-delineated
lymphoid follicles, no atypical mitosis, B-lymphocytes (anti-CD20, DAKO, Carpentería, CA,
USA) in the germinal center (Figure 2, panel A), T-lymphocytes (anti-CD45 RO+) in the
follicular zone (Figure 2, panel B), and no CD3+ cells.
DNA was extracted from the biopsy samples (Repli-g Mini Kit, QIAGEN, Valencia, CA,
USA) and used for PCR amplification of a fragment of fla gene specific for B. burgdorferi sensu
lato species as well as for a fragment of ospA gene, as described (8–10). DNA from a skin biopsy
of a patient with systemic lupus erythematous was used as negative control, and DNA (10
pg/μL) from B. burgdorferi sensu stricto B31 served as positive control. All procedures from
DNA extraction to amplification were performed twice for each sample. Amplified products
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were further tested by Southern blot (SB) hybridization with probes specific for B. burgdorferi
sensu stricto, B. garinii, and B. afzelii, as described (9). DNA from the 4 biopsy samples was
positive for B. burgdorferi sensu lato fla gene by PCR and confirmed as B. burgdorferi sensu
stricto by SB (Appendix Figure, Table). All DNA biopsy samples were negative by SB with the
probes specific for B. garinii and B. afzelii. We were able to amplify the OspA gene for only 1
case of erythema migrans, by using PCR and SB tests (data not shown).
The PCR products of the fla gene from 3 patients and of the ospA gene from 1 patient
were sequenced by using a commercial kit (GenomeLab DTCS-Quick Start Kit, Beckman
Coulter, Inc., Fullerton, CA, USA) with the sequencer from Beckman Coulter, Inc., according to
manufacturer’s instructions. We used the DNAMAN program (Lynnon Corporation, Vaudreuil-
Dorion, Quebec, Canada) to align the sequences with the reported sequences of the B.
burgdorferi sensu stricto B31 strain (online Appendix Figure). For the 2 erythema migrans cases,
we found 3 base substitutions Appendix Figure, panel C), 1 of which was not conserved, leading
to a change in amino acid (G for R in the 75 aa); these 2 sequences had 99% homology with the
sequence of B. burgdorferi fla gene of isolate B31 (BLAST program) (11). For the
lymphocytoma case, we found 2 base substitutions, the same as those of the erythema migrans
cases, including the nonconserved base substitution (Appendix Figure, panel C).
Regarding the ospA gene in the erythema migrans case, the sequence showed 1 base
substitution that was not conserved, leading to a change in the amino acid 5 (L for I). The
sequence of this case had 99% homology with the plasmid Ip54 gene of B31strain sequence (11)
(Appendix Figure, panel D).
The 3 adult patients received doxycycline 200 mg/day for 3 weeks; the child received
amoxicillin 50 mg/kg a day for 3 weeks. For all patients, lesions were gone at the end of the
treatment and had not recurred 3 years later.
Conclusions
Erythema migrans is the diagnostic marker for Lyme disease associated with
B. burgdorferi infection (5,6). Histologic data from our 2 erythema migrans cases agreed with
data reported for other erythema migrans cases (5). Moreover, the 2 erythema migrans cases
were positive for B. burgdorferi sensu stricto fla gene and 1 for ospA gene; the 3 cases had a high
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degree of homology to the sequences of strain B31. In addition, 1 case met CDC criteria for
seropositivity to B. burgdorferi infection (5).
Borrelial lymphocytoma is a rare clinical entity reported mostly in Europe (12–14) and
sporadically in the United States (15). In this study, histologic and immunohistochemical data
from the 2 lymphocytoma cases agreed with data from previous cases. These results were not
specific enough to be considered diagnostic; however, germinal centers are present in 80% of
borrelial lymphocytoma cases (12). Serum samples from 2 patients were positive by WB, which
fulfills CDC criteria (5). In 1 case, fla gene was amplified and sequenced, showing high
homology with the fla gene from B. burgdorferi sensu stricto strain B31 (11). Few reports
describe genotyping of B. burgdorferi species in borrelial lymphocytoma. In Slovenia, B. afzelii
and B. bissettii were identified (13); in Germany, B. garinii was identified (14). In our
lymphocytoma patients, we identified B. burgdorferi sensu stricto. That the 2 borrelial
lymphocytoma cases occurred in patients who had visited the same national park suggests that
B. burgdorferi is endemic to that area.
This study documents B. burgdorferi infection in Mexican patients. Relevant
epidemiologic data are 1) cases occurred after visiting forest areas, 2) patients reported having
been bitten by a nonflying insect, 3) cases occurred during the summer-fall season, 4) no patient
reported having traveled to another country, and 5) all skin lesions resolved after treatment with
an antimicrobial drug. Our results suggest that B. burgdorferi infection occurs in Mexico and that
continuous surveillance for Lyme disease in Mexico should be mandatory.
Acknowledgments
We thank C. Barthel for her excellent technical assistance.
Financial support was provided by the Consejo Nacional de Ciencia y Tecnología, Mexico (grant 30694-M)
and by the Coordinación de Investigación en Salud, Instituto Mexicano del Seguro Social (IMSS), grant FP-
2003/119, México. J.T. has a Fundacion-IMSS exclusivity scholarship.
Dr Gordillo-Pérez is an associate researcher in the Infectious Diseases Research Unit at the Paediatric
Hospital, Centro Médico Nacional Siglo XXI, IMSS, in Mexico City. Her research interests are diagnosis and
epidemiology of Lyme borreliosis in Mexico, molecular typing of B. burgdorferi and its vectors, and infections of
the central nervous system.

[Kritters]

Hey Lady,
Can you give link for this?
Kritts

[ladycolorado]

kritters sure, yes just adobe acrobat, i hate it, lol no problem: http://www.cdc.gov/eid/content/13/10/pdfs/06-0630.pdf . here is the ahead of print page on cdc emerging infectious diseases, cool did not know til today could see ahead of print found by accident: http://www.cdc.gov/ncidod/eid/upcoming.htm . (LC).

[Kritters]

thanks, L.C.
xoxo
Kritts