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    Inoculation eschar at the tick bite site on the left ankle of a patient infected with Rickettsia sp. strain Atlantic rainforest in Santa Catarina State, southern Brazil. (A) 12 days after the tick bite (DATB). (B) 14 DATB. (C) 23 DATB. (D) 30 DATB.

  • 1.

    Spolidorio MG, Labruna MB, Mantovani E, Brandão P, Richtzenhain LJ, Yoshinari NH, 2010. Novel spotted fever group rickettsioses, Brazil. Emerg Infect Dis 16: 521523.

    • Search Google Scholar
    • Export Citation
  • 2.

    Silva N, Eremeeva ME, Rozental T, Ribeiro GS, Paddock CD, Ramos EAG, Favacho ARM, Reis MG, Dasch GA, de Lemos ERS, Ko AI, 2011. Eschar-associated spotted fever rickettsiosis, Bahia, Brazil. Emerg Infect Dis 17: 275278.

    • Search Google Scholar
    • Export Citation
  • 3.

    Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D, 2013. Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev 26: 657702.

    • Search Google Scholar
    • Export Citation
  • 4.

    Nieri-Bastos FA, Horta MC, Barros-Battesti DM, Moraes-Filho J, Ramirez DG, Martins TF, Labruna MB, 2016. Isolation of the pathogen Rickettsia sp. strain Atlantic rainforest from its presumed tick vector, Amblyomma ovale (Acari: Ixodidae), from two areas of Brazil. J Med Entomol pii: tjw062.

    • Search Google Scholar
    • Export Citation
  • 5.

    Paddock CD, Finley RW, Wright CS, Robinson HN, Schrodt BJ, Lane CC, Ekenna O, Blass MA, Tamminga CL, Ohl CA, McLellan SL, Goddard J, Holman RC, Openshaw JJ, Sumner JW, Zaki SR, Eremeeva ME, 2008. Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever. Clin Infect Dis 47: 11881196.

    • Search Google Scholar
    • Export Citation
  • 6.

    Conti-Díaz IA, Moraes-Filho J, Pacheco RC, Labruna MB, 2009. Serological evidence of Rickettsia parkeri as the etiological agent of rickettsiosis in Uruguay. Rev Inst Med Trop Sao Paulo 51: 337339.

    • Search Google Scholar
    • Export Citation
  • 7.

    Romer Y, Nava S, Govedic F, Cicuttin G, Denison AM, Singleton J, Kelly AJ, Kato CY, Paddock CD, 2014. Rickettsia parkeri rickettsiosis in different ecological regions of Argentina and its association with Amblyomma tigrinum as a potential vector. Am J Trop Med Hyg 91: 11561160.

    • Search Google Scholar
    • Export Citation
  • 8.

    Angerami RN, da Silva AM, Nascimento EM, Colombo S, Wada MY, dos Santos FC, Mancini DM, de Oliveira RC, Katz G, Martins EC, da Silva LJ, 2009. Brazilian spotted fever: two faces of a same disease? A comparative study of clinical aspects between an old and a new endemic area in Brazil. Clin Microbiol Infect 2 (Suppl): 207208.

    • Search Google Scholar
    • Export Citation
  • 9.

    Barros e Silva PMR, Pereira SC, Fonseca LX, Maniglia FVP, Oliveira SV, de Caldas EP, 2014. Febre maculosa: uma análise epidemiológica dos registros dosistema de vigilância do Brasil. Scientia Plena 10: 19.

    • Search Google Scholar
    • Export Citation
  • 10.

    Barbieri ARM, Moraes-Filho J, Nieri-Bastos FA, Souza JC, Szabó MPJ, Labruna MB, 2014. Epidemiology of Rickettsia sp. strain Atlantic rainforest in a spotted fever-endemic area of southern Brazil. Ticks Tick Borne Dis 5: 848853.

    • Search Google Scholar
    • Export Citation
  • 11.

    Labruna MB, Horta MC, Aguiar DM, Cavalcante GT, Pinter A, Gennari SM, Camargo LMA, 2007. Prevalence of Rickettsia infection in dogs from the urban and rural areas of Monte Negro Municipality, western Amazon, Brazil. Vector Borne Zoonotic Dis 7: 249255.

    • Search Google Scholar
    • Export Citation
  • 12.

    Eremeeva ME, Bosserman EA, Demma LJ, Zambrano ML, Blau DM, Dasch GA, 2006. Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona. Appl Environ Microbiol 72: 55695577.

    • Search Google Scholar
    • Export Citation
  • 13.

    Roux V, Raoult D, 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 50: 14491455.

    • Search Google Scholar
    • Export Citation
  • 14.

    Medeiros AP, de Souza AP, de Moura AB, Lavina MS, Belatto V, Sartor AA, Nieri-Bastos FA, Richtzenhain LJ, Labruna MB, 2011. Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil. Mem Inst Oswaldo Cruz 106: 926930.

    • Search Google Scholar
    • Export Citation
  • 15.

    Krawczak FS, Agostinho WC, Polo G, Moraes-Filho J, Labruna MB, 2016. Comparative evaluation of Amblyomma ovale ticks infected and noninfected by Rickettsia sp. strain Atlantic rainforest, the agent of an emerging rickettsiosis in Brazil. Ticks Tick Borne Dis 7: 502507.

    • Search Google Scholar
    • Export Citation
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Rickettsia sp. Strain Atlantic Rainforest Infection in a Patient from a Spotted Fever-Endemic Area in Southern Brazil

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  • 1 Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazil.
  • | 2 Fundação Universidade Regional de Blumenau, Blumenau, Santa Catarina, Brazil.
  • | 3 Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasília, Distrito Federal, Brazil.
  • | 4 Instituto Adolfo Lutz, Secretaria de Estado da Saúde, São Paulo, Brazil.
  • | 5 Hospital das Clínicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.

Santa Catarina State in southern Brazil is the state with the second highest number of laboratory-confirmed cases of spotted fever illness in Brazil. However, all these cases were confirmed solely by serological analysis (seroconversion to spotted fever group rickettsiae), which has not allowed identification of the rickettsial agent. Here, a clinical case of spotted fever illness from Santa Catarina is shown by seroconversion and molecular analysis to be caused by Rickettsia sp. strain Atlantic rainforest. This is the third confirmed clinical case due to this emerging rickettsial agent in Brazil. Like the previous two cases, the patient presented an inoculation eschar at the tick bite site. Our molecular diagnosis was performed on DNA extracted from the crust removed from the eschar. These results are supported by previous epidemiological studies in Santa Catarina, which showed that nearly 10% of the most common human-biting ticks were infected by Rickettsia sp. strain Atlantic rainforest.

Introduction

Until 2009, Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF) or Brazilian spotted fever (BSF), was the only known tick-borne pathogenic Rickettsia known to occur in Brazil. In 2010 in the state of São Paulo, southeastern Brazil, a tick-borne spotted fever illness was reported to be caused by a novel rickettsial agent genetically related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. This novel agent was named Rickettsia sp. strain Atlantic rainforest in allusion to the forest environment where the patient presumably acquired the infected tick.1 In the subsequent year, another case of tick-borne spotted fever illness caused by a novel rickettsial agent was reported in the state of Bahia, northeastern Brazil.2 Although the rickettsial agent of this second case was named Rickettsia sp. strain Bahia, further studies considered it to be the second clinical case of Rickettsia sp. strain Atlantic rainforest, since the two strains share identical DNA sequences.3,4 These two cases were milder than the classical R. rickettsii-caused BSF cases from southeastern Brazil, and more interestingly, presented inoculation eschar (tache noire) at the tick bite site, a lesion typically described in cases of R. parkeri rickettsiosis in Argentina, Uruguay, and the United States, but usually not for RMSF or BSF.57

Between 2005 and 2015, a total of 314 laboratory-confirmed cases of spotted fever rickettsiosis were officially reported for the state of Santa Catarina, southern Brazil. All these cases have been confirmed by seroconversion to R. rickettsii antigens through analyses of acute and convalescent serum samples (unpublished official data from the Brazilian Ministry of Health). In the whole country, only São Paulo State reported more spotted fever cases than Santa Catarina; however, whereas BSF fatality rates have been around 40% in São Paulo State, no fatality has been observed in Santa Catarina.8,9 These intriguing differences have led to suspect that the spotted fever cases of Santa Catarina were caused by a tick-borne agent different from R. rickettsii.8 A recent epidemiological study suggested that at least part of the spotted fever cases of Santa Catarina was caused by Rickettsia sp. strain Atlantic rainforest, based on the detection of this agent in nearly 10% of the ticks collected from domestic dogs in the area where most of clinical spotted fever cases were reported.10 Regardless, no rickettsial agent has been determined for the spotted fever cases of Santa Catarina.

Case Report

In January 28, 2015, a 21-year-old woman (biology undergraduate student) was bitten by a tick on her left ankle during an ornithological expedition in a degraded fragment of Atlantic rainforest (26°51′24″S, 49°03′06″W; 21 m), Blumenau Municipality, Santa Catarina State, southern Brazil. Unfortunately, the tick was discarded after removal from the skin. Seven days after the tick bite (February 4, 2015), the patient reported a papular skin lesion at the tick bite site with intense pruritus. On February 7 (10 days after the tick bite), the patient reported headache associated with myalgia and arthralgia. On the next day, the papular lesion became surrounded by a macular rash, and symptoms of chills and fever were reported. On February 9, the patient visited a physician, who prescribed paracetamol and oral doxycycline (100 mg, every 12 hours) for 14 days. At this moment, the patient presented fever (38°C), muscle and joint pain. While fever and general pain resolved 5 days after, the skin lesion at the tick bite site evolved to a lesion with a necrotic center (inoculation eschar or tache noire), which was nearly completely healed at February 27, 30 days after the tick bite (Figure 1). Disseminated rash was not observed.

Figure 1.
Figure 1.

Inoculation eschar at the tick bite site on the left ankle of a patient infected with Rickettsia sp. strain Atlantic rainforest in Santa Catarina State, southern Brazil. (A) 12 days after the tick bite (DATB). (B) 14 DATB. (C) 23 DATB. (D) 30 DATB.

Citation: The American Society of Tropical Medicine and Hygiene 95, 3; 10.4269/ajtmh.16-0192

Because of suspicion of tick-borne rickettsial disease, the case was notified to the official health system of Brazil, where spotted fever is an obligate notifiable disease. Serum samples were collected at the day of the first medical visit (February 9, 2015), and 8 days later (20 days after the tick bite). The paired serum samples were tested by immunofluorescence antibody assay against crude antigens of six Rickettsia species from Brazil (Rickettsia bellii, Rickettsia felis, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia rickettsii, and R. parkeri), as previously described.10,11 Briefly, sera were diluted in 2-fold increments with phosphate-buffered saline, starting from the 1:64 dilution. Slides were incubated with fluorescein isothiocyanate–labeled goat antihuman IgG (Sigma, St. Louis, MO). For each sample, the end point IgG titer reacting with each of the six Rickettsia antigens was determined. Although the first serum sample did not react to any rickettsial antigen (end point titers < 64), the second serum sample had end point titers of 1,024 to R. rickettsii, R. parkeri, and R. amblyommii, 512 to R. rhipicephali and R. bellii, and < 64 to R. felis. These results indicate a seroconversion with at least 16-fold increase in titers of antibodies against R. parkeri, R. rickettsii, and R. amblyommii.

Twelve days after the tick bite (February 9, 2015), the patient removed the crust of the wound at the tick bite site (Figure 1) and stored it in a sterile microtube with 96% ethanol. Four months later, DNA of this skin sample was extracted using the DNAeasy Blood and Tissue Kit (Qiagen, Valencia, CA), and tested by polymerase chain reaction (PCR) with the primers Rr190.70F and Rr190.701R, targeting a ∼630-bp fragment of the rickettsial ompA gene,12 and by the primers 120-M59 and 120-807, targeting a ∼820-bp fragment of the rickettsial ompB gene.13 PCR products were purified with ExoSap (USB, Cleveland, OH) and sequenced in an ABI Prism 310 Genetic Analyzer (Applied Biosystems/Perkin Elmer, Foster City, CA). Generated sequences were shown by Basic Local Alignment Search Tool analyses (www.ncbi.nlm.nih.gov/blast) to be 100% identical to ompA (JQ906784) and ompB (GQ855236) corresponding sequences of Rickettsia sp. strain Atlantic rainforest. Partial DNA sequences of ompA (590-bp) and ompB (817-bp) genes generated in this study were deposited in GenBank under accession numbers KU882101 and KU882102.

Discussion

A clinical case of spotted fever is confirmed by molecular analysis for the first time in southern Brazil. This is the third confirmed case of human illness due to Rickettsia sp. strain Atlantic rainforest. Our results are supported by previous epidemiological studies in Santa Catarina, which showed that nearly 10% of the ticks Amblyomma ovale and Amblyomma aureolatum were infected by Rickettsia sp. strain Atlantic rainforest, and that these two tick species were the most common human-biting ticks in the areas with reported human cases of rickettsiosis in Santa Catarina.10,14 In addition, a recent laboratory study demonstrated the vector competence of A. ovale for Rickettsia sp. strain Atlantic rainforest.15

Before this study, all previous spotted fever cases in southern Brazil, including the area of this study, were confirmed solely by serological analysis (seroconversion to R. rickettsii antigens),8,10 which does not allow a precise identification of the rickettsial agent, since serologic cross-reaction usually occurs between different spotted fever group Rickettsia species.3 In fact, while our molecular analysis identified Rickettsia sp. strain Atlantic rainforest as the etiological agent, the patient seroconverted with identical end point titers to three spotted fever group agents, R. parkeri, R. rickettsii, and R. amblyommii. Similarly, the first report of human illness due to Rickettsia sp. strain Atlantic rainforest also resulted in seroconversion with similar end point titers to these three Rickettsia species.1 Because previous spotted fever clinical cases of Santa Catarina8,10 are clinically, serologically, and epidemiologically compatible with the three reported cases of Rickettsia sp. strain Atlantic rainforest (present study1,2), it is likely that this rickettsial agent is an important cause of spotted fever in southern Brazil.

Indeed, there is an urgent need for improvement of both epidemiological surveillance strategies and laboratory capacity to detect and differentiate other rickettsiosis, including mild or atypical spotted fever cases previously attributed to R. rickettsii based on serologic assays and not by direct tests. Here we pointed out the simplicity of collection and preservation of viable biological samples to be tested by PCR for detection of Rickettsia sp. strain Atlantic rainforest and other potentially pathogenic but not usually recognized Rickettsia species. Finally, because Rickettsia sp. strain Atlantic rainforest-infected A. ovale ticks seem to have a wide distribution in Latin America,4 the adoption of more specific diagnostic methods could result in more human cases due to this emerging rickettsial agent in different areas of the continent.

  • 1.

    Spolidorio MG, Labruna MB, Mantovani E, Brandão P, Richtzenhain LJ, Yoshinari NH, 2010. Novel spotted fever group rickettsioses, Brazil. Emerg Infect Dis 16: 521523.

    • Search Google Scholar
    • Export Citation
  • 2.

    Silva N, Eremeeva ME, Rozental T, Ribeiro GS, Paddock CD, Ramos EAG, Favacho ARM, Reis MG, Dasch GA, de Lemos ERS, Ko AI, 2011. Eschar-associated spotted fever rickettsiosis, Bahia, Brazil. Emerg Infect Dis 17: 275278.

    • Search Google Scholar
    • Export Citation
  • 3.

    Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D, 2013. Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev 26: 657702.

    • Search Google Scholar
    • Export Citation
  • 4.

    Nieri-Bastos FA, Horta MC, Barros-Battesti DM, Moraes-Filho J, Ramirez DG, Martins TF, Labruna MB, 2016. Isolation of the pathogen Rickettsia sp. strain Atlantic rainforest from its presumed tick vector, Amblyomma ovale (Acari: Ixodidae), from two areas of Brazil. J Med Entomol pii: tjw062.

    • Search Google Scholar
    • Export Citation
  • 5.

    Paddock CD, Finley RW, Wright CS, Robinson HN, Schrodt BJ, Lane CC, Ekenna O, Blass MA, Tamminga CL, Ohl CA, McLellan SL, Goddard J, Holman RC, Openshaw JJ, Sumner JW, Zaki SR, Eremeeva ME, 2008. Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever. Clin Infect Dis 47: 11881196.

    • Search Google Scholar
    • Export Citation
  • 6.

    Conti-Díaz IA, Moraes-Filho J, Pacheco RC, Labruna MB, 2009. Serological evidence of Rickettsia parkeri as the etiological agent of rickettsiosis in Uruguay. Rev Inst Med Trop Sao Paulo 51: 337339.

    • Search Google Scholar
    • Export Citation
  • 7.

    Romer Y, Nava S, Govedic F, Cicuttin G, Denison AM, Singleton J, Kelly AJ, Kato CY, Paddock CD, 2014. Rickettsia parkeri rickettsiosis in different ecological regions of Argentina and its association with Amblyomma tigrinum as a potential vector. Am J Trop Med Hyg 91: 11561160.

    • Search Google Scholar
    • Export Citation
  • 8.

    Angerami RN, da Silva AM, Nascimento EM, Colombo S, Wada MY, dos Santos FC, Mancini DM, de Oliveira RC, Katz G, Martins EC, da Silva LJ, 2009. Brazilian spotted fever: two faces of a same disease? A comparative study of clinical aspects between an old and a new endemic area in Brazil. Clin Microbiol Infect 2 (Suppl): 207208.

    • Search Google Scholar
    • Export Citation
  • 9.

    Barros e Silva PMR, Pereira SC, Fonseca LX, Maniglia FVP, Oliveira SV, de Caldas EP, 2014. Febre maculosa: uma análise epidemiológica dos registros dosistema de vigilância do Brasil. Scientia Plena 10: 19.

    • Search Google Scholar
    • Export Citation
  • 10.

    Barbieri ARM, Moraes-Filho J, Nieri-Bastos FA, Souza JC, Szabó MPJ, Labruna MB, 2014. Epidemiology of Rickettsia sp. strain Atlantic rainforest in a spotted fever-endemic area of southern Brazil. Ticks Tick Borne Dis 5: 848853.

    • Search Google Scholar
    • Export Citation
  • 11.

    Labruna MB, Horta MC, Aguiar DM, Cavalcante GT, Pinter A, Gennari SM, Camargo LMA, 2007. Prevalence of Rickettsia infection in dogs from the urban and rural areas of Monte Negro Municipality, western Amazon, Brazil. Vector Borne Zoonotic Dis 7: 249255.

    • Search Google Scholar
    • Export Citation
  • 12.

    Eremeeva ME, Bosserman EA, Demma LJ, Zambrano ML, Blau DM, Dasch GA, 2006. Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona. Appl Environ Microbiol 72: 55695577.

    • Search Google Scholar
    • Export Citation
  • 13.

    Roux V, Raoult D, 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 50: 14491455.

    • Search Google Scholar
    • Export Citation
  • 14.

    Medeiros AP, de Souza AP, de Moura AB, Lavina MS, Belatto V, Sartor AA, Nieri-Bastos FA, Richtzenhain LJ, Labruna MB, 2011. Spotted fever group Rickettsia infecting ticks (Acari: Ixodidae) in the state of Santa Catarina, Brazil. Mem Inst Oswaldo Cruz 106: 926930.

    • Search Google Scholar
    • Export Citation
  • 15.

    Krawczak FS, Agostinho WC, Polo G, Moraes-Filho J, Labruna MB, 2016. Comparative evaluation of Amblyomma ovale ticks infected and noninfected by Rickettsia sp. strain Atlantic rainforest, the agent of an emerging rickettsiosis in Brazil. Ticks Tick Borne Dis 7: 502507.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Marcelo B. Labruna, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Avenue Professor Orlando Marques de Paiva, 87 Cidade Universitária, São Paulo, 05508-270, Brazil. E-mail: labruna@usp.br

Financial support: This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Authors' addresses: Felipe S. Krawczak, Sebastián Muñoz-Leal, Jonas Moraes-Filho, and Marcelo B. Labruna, Department of Preventive Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil, E-mails: felipekvet@gmail.com, seba.munozleal@gmail.com, jonasmfilho@hotmail.com, and labruna@usp.br. Ana Carolina Guztzazky and Julio C. de Souza Jr., Biologia, Universidade Regional de Blumenau, Blumenau, Brazil, E-mails: anacarolina.gmariana@gmail.com and juliocesar_jr@terra.com.br. Stefan V. Oliveira, Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasilia, Distrito Federal, Brazil, E-mail: stefanbio@yahoo.com.br. Fabiana C. P. Santos, Nucelo de Anatomica Patologica, Instituto Adolfo Lutz, Sao Paulo, Brazil, E-mail: fabianacp@yahoo.com. Rodrigo N. Angerami, Departamento de Vigilancia em Saude, Secretaria Municipal de Saude de Campinas, Campinas, Sao Paulo, Brazil, E-mail: rodrigo.angerami@gmail.com.

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