REPORT

 

Evidences of autochthonous infection by Borrelia burgdorferi sensu lato in Cuba

 

Evidencias de infección autóctona por Borrelia burgdorferi sensu lato en Cuba

 

 

Islay Rodríguez¹, Carmen Fernández¹, Omar Fuentes¹, Roberto A González², Bárbara Martínez³, Eduardo Echevarría¹, Ángel A Noda¹, Ana M Obregón¹

¹ Instituto de Medicina Tropical Pedro Kourí, IPK. Autopista Novia del Mediodía, Km 6½, La Lisa, CP 11 400, La Habana, Cuba.
² Unidad de Control de Vectores, Sierra del Rosario, Artemisa, Cuba.
³ Centro de Asistencia Médica, Sierra del Rosario, Artemisa, Cuba.

 

 

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ABSTRACT

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, borreliosis not reported in Cuba but clinical and epidemiological suspicions exist since the 1980’s, and there were no microbiological testing for confirmation. This led to perform a series of researches to provide scientific evidences about the presence of this agent in the country, including the evaluation and implementation of microbiological methods for the detection of this spirochete, confirmation of infection in clinical samples from patients with clinical and epidemiological suspicions, the estimated seroprevalence of antibodies against this agent in a population at risk and the molecular detection of borrelia in ticks of veterinary and medical importance. The microbiological tools evaluated (culture medium modified, methods of extracting genetic material from ticks for subsequent detection of B. burgdorferi sensu lato by molecular testing, and analytical sensitivity of two sets of primers) and implemented (specific serological tests) strengthened the IPK analytical capacity with novel methods for future researches. We found specific serological evidences of autochthonous infection by this organism in samples of individuals with clinical and epidemiological suspicion, and those exposed to tick bites. Genetic material of B. burgdorferi sensu lato was undetected in the analyzed ticks, what does not rule them out as potential carriers of the agent. In this paper we show evidence highly suggestive of infection with B. burgdorferi sensu lato and it is the first of its kind in the country.

Keywords: Borrelia burgdorferi, Borrelia, infection, tick, Cuba.

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RESUMEN

Borrelia burgdorferi sensu lato causa la enfermedad de Lyme, una borreliosis no reportada en Cuba. Desde la década de 1980 se tenían sospechas clínicas y epidemiológicas, pero no había pruebas microbiológicas que lo confirmaran. Las investigaciones científicas sobre su presencia en el país incluyeron la evaluación e implementación de métodos microbiológicos para su detección, la confirmación de la infección en muestras clínicas de pacientes con sospechas clínico-epidemiológicas de esta enfermedad, la estimación de la seroprevalencia de anticuerpos contra este agente en una población de riesgo y la detección molecular de borrelias en garrapatas de importancia médico-veterinaria. Las herramientas microbiológicas evaluadas (medio de cultivo modificado, métodos de extracción de material genético de garrapatas para la posterior detección de B. burgdorferi sensu lato por pruebas moleculares, y la sensibilidad analítica de dos juegos de cebadores) e implementadas (pruebas serológicas específicas) fortalecieron la capacidad analítica del Instituto de Medicina Tropical Pedro Kourí, en Cuba, con nuevos métodos para futuras investigaciones. En las muestras de individuos con sospechas clínico-epidemiológicas y en los expuestos a mordeduras por garrapatas, se encontraron evidencias serológicas específicas de la infección autóctona por este microorganismo. No se detectó material genético de B. burgdorferi sensu lato en las garrapatas analizadas, lo que no descarta que sean posibles vectores del agente. Las evidencias de este estudio son altamente sugestivas y constituye el primero de su tipo en Cuba.

Palabras clave: Borrelia burgdorferi, Borrelia, infección, garrapata, Cuba.

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INTRODUCTION

Borrelia burgdorferi sensu lato is a complex of spirochetes [1], which cause Lyme disease, zoonosis for which small and large mammals are the main reservoirs of Borrelia, which is spread mainly by hard ticks of the genus Ixodes [2]. By the late twentieth century, this entity was recognized as an important emerging disease by severe consequences to human health and the difficulties for its prevention and control [3]. This disease has only been described in the northern hemisphere and is the most common vector-borne illness in the United States of America and Eurasia regions [2].

In Cuba, since the 1980’s, it is suspected the occurrence of infection with B. burgdorferi sensu lato in a community located in Sierra del Rosario, Candelaria, Artemisa province. The tick infestation in this place is high, mainly due to Amblyomma cajennense, and among the human population are reported historically idiopathic syndromes with a clinical and epidemiological background that mimics Lyme disease, but could not be confirmed by absence of laboratory diagnosis [4].

The first evidence of a probable case of infection with B. burgdorferi sensu lato also dates from the 1980s, an individual who was diagnosed retrospectively by a screening test as neuroborreliosis in a foreign country [5].

This serological finding in a Cuban individual reinforced the suspicion of the existence of infection with B. burgdorferi sensu lato in Cuba; so that it became necessary to initiate microbiological investigations to provide scientific evidence about it.

For that, it was combined a group of studies with different epidemiological designs. Three experimental studies were conducted to evaluate microbiological methods of detection of B. burgdorferi sensu lato: a) evaluation of a modified culture medium for multiplication of B. burgdorferi sensu lato, b) evaluation of methods for extracting genetic material from ticks, for later use in the detection of this agent by PCR, and c) evaluation of the analytical sensitivity of PCR for detection of this spirochete. Two case-series studies were conducted using clinical samples of individuals with clinical and epidemiologic suspicion, divided into two periods (1998-2002 and 2003-2010) for confirmation of infection with B. burgdorferi sensu lato in humans. A cross-sectional observational study was performed from January to May 2006 to estimate the seroprevalence of antibodies against B. burgdorferi sensu stricto in a population at risk. In addition, two observational studies were conducted in different reservoirs: a) ticks collected from cattle and horses, and b) ticks collected from cattle, dogs and a house for the detection of Borrelia in ticks.

 

RESULTS AND DISCUSSION

Microbiological evaluation of methods for the detection of B. burgdorferi sensu lato

Evaluation of a modified culture medium for B. burgdorferi sensu lato

The modified medium was prepared according to the composition of BSK-II (commercial name: BSK-H) [6] replacing the medium CMRL 1066 by the medium 199 for not having it.

Strains NE4546 and NE83 of B. garinii, NE4502 of B. burgdorferi sensu stricto, NE4505 of B. afzelii and NE4555 of B. valaisiana were used to conduct a qualitative and quantitative evaluation of it. The commercial medium was used as control.

In the qualitative evaluation, we observed a color change from pink to orange in both media due to a decrease of pH as a consequence of the onset and increased the concentration of the final products of fermentation in media. Using the dark field microscopy, uniformly refractile and mobile spirochetes in both media were observed.

The quantitative evaluation yielded the growth of B. burgdorferi sensu stricto and B. garinii strains in both media. The multiplication of B. afzelii strain in the modified medium was not possible, only some borrelies with reduced motility were observed. It is reported that different strains of Borrelia species grow unevenly [7].

In addition, the analysis of the protein profile of the strain NE83 of B. garinii grown in these media and their antigenic properties of proteins was performed, which revealed the expression of the main proteins, both specific and nonspecific, and that a large number of these were antigenic, whatever the medium used. These results suggest that the modified medium evaluated can be used as an alternative to the cultivation of Borrelia strains in countries with few resources, taking into account its limitations for the growth of certain strains.

Evaluation of the efficacy of extraction methods for nucleic acids from hard ticks

From May to October 2007, the following protocols for DNA extraction were evaluated using the tick Rhipicephalus (Boophilus) microplus: the phenol-chloroform extraction proposed by Weng et al. [8], Halos et al. [9] and Fraga et al. [10] with modifications; the potassium acetate method reported by Gaillard and Strauss [11] with some modifications and the protocol of extraction using ammonium hydroxide and heat modified by Humair et al. [12].

Genomic DNA was only observed in extracts of adult female ticks obtained by Halos et al. [9] and Fraga et al. [10] procedures. RNA was confirmed in all of them, regardless of life stage and gender. PCR amplification of a gene fragment coding for the 16S subunit of mitochondrial RNA ticks allowed the identification of the DNA fragment in all cases except for the extracts obtained by the method of ammonium hydroxide and heat [10] in engorged female ticks. These results demonstrated the effectiveness of each procedure. This work constitutes the first report worldwide of the nucleic acid extraction using the potassium acetate method in ticks.

Evaluation of the analytical sensitivity of PCR to amplify DNA fragments of B. burgdorferi sensu lato

The evaluation by PCR of the 5S-23S intergenic region [13] and 16S subunit sequence [14] of B. burgdorferi sensu lato rRNA was done with reference DNA from B. burgdorferi sensu stricto (B31), B. afzelii (NE632), B. garinii (NE110 and B. lusitaniae (Poti-B1). The expected fragments for each of them were amplified. PCR of the 5S-23S intergenic region was able to detect 0.21 pg of DNA (84 copies of the genome), while PCR of the 16S sequence detected only 2.1 ng (8400 copies of the genome). Based on these results, it is proposed the PCR of 5S-23S for the detection of B. burgdorferi sensu lato.

Technology transfer of serological tests

The commercial system ELISA-C6 (IgM/IgG) (Immunetics, USA) and a Western blot-IgG (home-made) were established at the National Microbiology Laboratory of the Institute of Tropical Medicine Pedro Kourí (IPK) as screening and confirmatory tests, respectively.

Microbiological diagnosis of infection with B. burgdorferi sensu lato in clinical samples of individuals with clinical and epidemiological suspicion

Case-series study 1

During the period 1998-2002, 14 serum samples were studied, from an equal number of individuals with clinical and epidemiological suspicion of Lyme disease from a community of Sierra del Rosario, Artemisa. It was used Enzignost Borreliosis ELISA IgM/IgG (Behring, Margurg, Germany) as screening method and Western blot IgM and IgG as confirmatory test (University of Trieste, Italy).

Five of the samples had positive values of IgM antibodies and one, limit values of both IgM and IgG antibodies by ELISA. Two of the six samples were positive to IgM by Western blot, and one of them showed a low titer of IgM antibodies to leptospirosis, which reaffirms the antigenic similarity between Leptospira spp. and the causative agent of infection in this patient.

One positive case was retrospectively investigated; being a male patient aged 16 with no previous history of travel outside the country, being suspected for Lyme disease from the age of five due to clinical manifestations and tick bite history.

Case-series study 2

It was conducted during 2003-2010 in collaboration with the Laboratory of Microbiology, ADMed, La-Chaux-de-Fonds, Switzerland. We studied 27 serum, 11 cerebrospinal fluids, one peripheral blood and one plasma samples from 30 individuals with clinical and epidemiological suspicions of this borreliosis from different provinces of Cuba. As serological screening tests were used the IgM/IgG ELISA VIDAS (BioMérieux, France), IgM-capture ELISA (IDEIA^TM Borrelia IgM; Dako Cytomation, USA) and IgM/IgG ELISA-C6 (C6 Lyme ELISA^TM) (Immunetics, USA). As confirmatory tests were used in-house IgM (B. garinii (NE83) as antigen) and IgG (B. burgdorferi sensu stricto (B31) and B. garinii (NE83) as antigens) Western blot procedures, and also the Western blot-IgG (Bioline Diagnostic, Italy). The blood sample was studied by culture in BSK-H and BSK modified medium and by PCR 5S-23S.

Specific IgM antibodies were detected in 10% (3/30) of the individuals, confirming the presence of infection in early stages, while in 6.6% (2/30) of individuals the detection of specific IgG confirmed the presence of infection in disseminated or advanced stage. The positive samples were subjected to serological tests for leptospirosis and syphilis, being negative.

These results corroborated serological findings reported in the previous series, and confirmed the presence of this infection in Cuba based on serological evidence. The lack of previous history of travels abroad demonstrated the autochthonous nature of infection cases.

Prevalence of antibodies to B. burgdorferi sensu stricto in individuals from a population exposed to tick bites

This study was conducted from January to May 2006 in a community of Sierra del Rosario. The population at risk comprised 980 individuals, a representative sample of 247 of them selected through simple random sampling. Serum was collected for each individual and performed by Western blot-IgG analysis, (Ag: B31 strain of B. burgdorferi sensu stricto). For the interpretation, we used three different criteria: a) Criteria from the Microbiology Laboratory, ADMed of La-Chaux-de-Fonds, Switzerland: scoring system in accordance to band’s specificity, b) Criteria of the Centers for Disease Control and Prevention (CDC, USA) [15] and c) Criteria of the CDC with the inclusion of OspA (31 kDa) and OspB (34 kDa) proteins [16].

The table below shows results on the prevalence of antibodies using different interpretation criteria. There is an overlap among the confidence intervals, so it was estimated a seroprevalence of 0.6-7.2% of specific antibodies against B. burgdorferi sensu stricto as a result of getting together the three intervals. In this study, there were no cross-reactivity with antibodies against Leptospira sp. and Treponema sp., and there are no other reports of Borrelia in the country.

The bands corresponding to 72, 60, 47 and 41 kDa proteins were revealed in more than 50% of the samples, although of weak intensity. In the samples identified as positive it was observed that 90/93, 72, 65/66, 60, 58, 56, 47 and 41 kDa proteins were significantly revealed and they corresponded to both highly specific and non-specific B. burgdorferi sensu lato proteins. Other revealed highly specific proteins were the 30 and 22/24 (OspC) kDa proteins, but few sera reacted with them.

Specific serological evidences of infection with B. burgdorferi sensu lato in samples of individuals from different provinces (Artemisa, Havana, Sancti Spíritus and Holguín) found in previous case-series studies and in this one latter suggest the circulation of the bacteria in the western, central and eastern regions of Cuba.

Molecular detection of B. burgdorferi sensu lato in ticks of medial and veterinary relevance

There were two observational studies, in which ticks were collected from different reservoirs (horses, cattle, dogs and a house) in the provinces of Artemisa and Havana by purposive sampling. The first was held in collaboration with the Laboratory of Ecoepidemiology of Parasites, Institute of Biology, University of Neuchâtel, Switzerland.

Taxonomic identification of ticks collected for the observational study 1 identified three species. In horses, 60% (57/95) of ticks were identified as Dermacentor (Anocentor) nitens, 37.9% (36/95) as A. cajennense and 2.1% (5/95) as R. microplus. This last specie corresponded the 100% (3/3) of the ticks captured in cattle. The observational study 2 showed that cattle were infected by R. microplus and dogs by R. sanguineus (100% in both cases). Ticks collected in the house were classified as R. sanguineus. Tick species identified in these studies did not correspond to known vectors for Lyme disease.

Genetic material of B. burgdorferi sensu lato was not detected in the extracts of any of them, by PCR amplification and reverse-line hybridization (study 1) [17] and PCR 5S-23S (study 2) [13].

Given that the number of ticks tested in this study is limited and not representative of the country, it is necessary to collect more ticks from pets, wild animals, migratory birds and vegetation in the area of Sierra del Rosario and to extend the research to other regions.

 

RELEVANCE OF THE STUDY

In this study, a modified culture medium was suggested as an alternative for the growth of B. burgdorferi sensu lato and a new internationally recognized methodology for the extraction of nucleic acids in ticks.

For the first time in Cuba, the microbiological tools required for the microbiological (bacteriological, serological and molecular) detection of infections with B. burgdorferi sensu lato have been implemented for human clinical specimens and the possible vectors, at the National Reference Laboratory of Microbiology of the IPK.

The first specific serological evidences of infection with B. burgdorferi sensu lato in individuals with clinical or epidemiological suspicion of Lyme disease in Cuba have been also shown. This investigation also demonstrated that the infection with B. burgdorferi sensu lato is not a local situation (western Cuba), having a wider geographical distribution.

The body of evidences about B. burgdorferi sensu lato infection suggests its autochthonous transmission in Cuba, which is an epidemiological alert for national authorities of veterinary and public health.

 

ACKNOWLEDGEMENTS

Special thanks to everyone who contributed to the completion of this work. To my collaborators: Hans H Siegrist, Reto Lienhard and Cloe Scheurer, fron the Microbiology Laboratory, ADMed of La-Chaux-de-Fonds, Switzerland; Lise Gern and Olivier Rais from the Laboratory of Ecoepidemiology of Parasites, Institute of Biology, University of Neuchâtel, Switzerland; Marina Cinco from the University of Trieste, Italy; Rodobaldo Pedroso from the Medical Assistant Center of Sierra del Rosario, Artemisa, Cuba; and Lizet Sánchez, Lilia M Ortega, María E Rodríguez, Rodobaldo Pedroso, José E Rodríguez, Brian A Mondeja, Jorge Fraga, Milagro Mayet, Yanisia Duarte, Gilda Toraño, Yaindrys Rodríguez, Yanais Valdés, Nadia M Rodríguez and Armando Fernández^† from the IPK. We also thank the Swiss Confederation by the financial supports (fellowships) offered to the first author for the training and execution of a part of the investigations comprised by this Award of the Cuban Academy of Sciences 2011.

 

REFERENCES

1. Stanek G, Reiter M. The expanding Lyme Borrelia complex--clinical significance of genomic species? Clin Microbiol Infect. 2011;17(4):487-93.

2. Gern L. Tiques et borréliose de Lyme en Suisse occidentale. Bull Soc Neuchateloise Scie Nat. 2004;127(1):5-21.

3. Steere AC. Lyme disease. N Engl J Med. 2001;345(2):115-25.

4. Grandío O, Fernández A, Fernández M, Valera R, Fuentes O, Pelegrino J. Informe preliminar sobre investigaciones realizadas en el poblado “Las Terrazas”, Sierra del Rosario, sobre la existencia de la enfermedad de Lyme. Rev Cubana Pediatr. 1988;60(5):773.

5. Dickinson F, Fuentes O, Fernández A. ¿Borreliosis de Lyme en Cuba? In: Comité Organizador, editores. Abstracts of the V Congreso Latinoamericano de Medicina Tropical; 1997 Mar 3-7. La Habana: Instituto de Medicina Tropical Pedro Kourí. p. 172-3.

6. Barbour AG. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984;57(4):521-5.

7. Ruzic-Sabljic E, Strle F. Comparison of growth of Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto in MKP and BSK-II medium. Int J Med Microbiol. 2004;294(6):407-12.

8. Wen B, Jian R, Zhang Y, Chen R. Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet. J Clin Microbiol. 2002;40(9):3286-90.

9. Halos L, Jamal T, Vial L, Maillard R, Suau A, Le Menach A, et al. Determination of an efficient and reliable method for DNA extraction from ticks. Vet Res. 2004;35(6):709-13.

10. Fraga J, Rodríguez J, Fuentes O, Castex M, Fernández-Calienes A. Comparación entre 5 métodos para la extracción de ADN de triatomíneos: su utilización en la técnica de ADN polimórfico amplificado al azar. Rev Cubana Med Trop. 2004;56(3):208-13.

11. Gaillard C, Strauss F. Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res. 1990;18(2):378.

12. Humair PF, Douet V, Moran Cadenas F, Schouls LM, Van De Pol I, Gern L. Molecular identification of bloodmeal source in Ixodes ricinus ticks using 12S rDNA as a genetic marker. J Med Entomol. 2007;44(5):869-80.

13. Postic D, Assous MV, Grimont PA, Baranton G. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol. 1994;44(4):743-52.

14. Marconi RT, Garon CF. Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol. 1992;30(11):2830-4.

15. Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep. 1995;44(31):590-1.

16. Hilton E, Devoti J, Sood S. Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease. J Clin Microbiol. 1996;34(6):1353-4.

17. Poupon MA, Lommano E, Humair PF, Douet V, Rais O, Schaad M, et al. Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland. Appl Environ Microbiol. 2006;72(1):976-9.

 

 

Islay Rodríguez. Instituto de Medicina Tropical Pedro Kourí, IPK. Autopista Novia del Mediodía, Km 6½, La Lisa, CP 11 400, La Habana, Cuba. E-mail: [e-mail].