Seroepidemiological survey to investigate Rickettsia rickettsii and Rickettsia parkeri in municipalities of the southeast BrazilABSTRACT
Spotted fever is a tick-borne rickettsiosis caused by several Rickettsia species—including R. rickettsii, R. parkeri, and others—with varying degrees of pathogenicity. Its nonspecific symptoms often lead to misdiagnosis such as dengue. This study investigated anti-R. rickettsii and R. parkeri antibodies in 152 patients with acute febrile illness who tested negative for dengue. Serological analysis using immunofluorescence assay found 29 reactive samples (19%) at a 1:64 dilution. Among them, 20.6% were male and 58.6% female, with an average age of 42.6 years. The average sample collection time totaled 14.6 days. Reactive samples included 13.1% for R. rickettsii and 5.9% for R. parkeri. These results suggest possible rickettsial infections in patients initially suspected of dengue.
KEYWORDS:
Spotted fever; Rickettsia; Tick; Zoonosis
INTRODUCTION
Tick-borne diseases are a global public health concern. Their etiologic agents, Rickettsia spp., are obligate intracellular gram-negative bacteria that infect endothelial cells, leading to vasculitis and systemic inflammatory responses1. Rickettsia rickettsii and Rickettsia parkeri cause spotted fever, with confirmed cases in Brazil. Its symptoms are often confused with dengue after an incubation period of 2 to 14 days2,3.
Spotted fever due to R. rickettsii may present high fever, rash, and multiple organ dysfunction, carrying high lethality if untreated12. In southeast Brazil and northern Parana State, Rickettsia rickettsii is transmitted by Amblyomma sculptum and Amblyomma aureolatum, particularly in the metropolitan area of Sao Paulo State. On the other hand, Rickettsia parkeri, especially the Atlantic Rainforest strain, is associated with Amblyomma ovale in the Atlantic Forest and is linked to milder forms of the disease in Brazil3,4.
In the case of Amblyomma sculptum, human infections are more commonly associated with the immature stages of the species (larvae and nymphs), whereas adult Amblyomma aureolatum and Amblyomma ovale are primarily responsible for transmission5.
From 2007 to 2021, Brazil recorded 36,497 spotted fever notifications, averaging 170 confirmed cases per year6. Minas Gerais State ranks third in confirmed cases and second in fatalities, with a 32.8% lethality rate from 2007 to 2019, mostly affecting males aged 30–59 years with contact history involving ticks, capybaras, or domestic animals7.
Diagnosis relies on indirect immunofluorescence assay (IFA), immunohistochemistry, or polymerase chain reaction but due to the rapid progression of the disease, treatment should begin based on clinical-epidemiological suspicion before lab confirmation8,9.
Due to its nonspecific clinical presentation, especially in mild or oligosymptomatic cases, spotted fever is often misdiagnosed as more prevalent illnesses such as dengue10. Therefore, this study investigates the seroprevalence of antibodies to R. rickettsii and R. parkeri in the Triangulo Mineiro region of Minas Gerais State, targeting patients with acute febrile illness who tested negative for dengue.
MATERIAL AND METHODS
Epidemiological data from 4,124 patients who were referred to the Laboratory of Immunoparasitology Dr. Mario Endsfeldz Camargo at the Federal University of Uberlandia for dengue screening in 2019 were included in this study. Via convenience sampling, 1,856 aliquots of blood serum with negative results for dengue were selected, of which 152 were subjected to serological analysis for Rickettsia rickettsii and Rickettsia parkeri. No active recruitment occurred; only anonymized secondary data were used.
Selection criteria prioritized samples from rural/peri-urban areas or outdoor workers (52) and those collected ≥14 days after symptom onset (100) to increase likelihood of Rickettsia exposure and IgG detection. Samples with missing epidemiological data or unsuitable storage conditions (hemolysis, lipemia, or low volume) were excluded. All selected samples were dengue-negative by IgM ELISA (PanBio™, Abbott).
Serum was tested by in-house IFA using Brazilian strains of R. rickettsii and R. parkeri that were provided by Prof. Marcelo B. Labruna from the Faculty of Veterinary Medicine and Zootechny of the University of Sao Paulo. The analyses were conducted at the Ixodology Laboratory of the Faculty of Veterinary Medicine at the Federal University of Uberlandia.
Sera were screened at 1:64 dilution using FITC-labeled anti-human IgG (1:600, Sigma), with positive and negative controls. Reactive samples were titrated in twofold dilutions and analyzed by epifluorescence microscopy at 40×.
Ethics
This study was approved by the UFU Research Ethics Committee (process Nº 5.392.382) and complied with National Research Ethics Commission guidelines (Resolutions Nº 466, of December 12, 2012, and Nº 510, of April 7, 2016, of the National Health Council).
RESULTS
Of the 152 analyzed samples, 29 (19%) were reactive at 1:64 dilution for R. rickettsii or R. parkeri. No reactivity occurred at higher titers. Most were female (58.6%), with an average age of 42.6 years and collection time of 14.6 days. Of the reactive cases, 41.3% were White, 27.6% Brown, and 24.1% Black individuals, mainly from Uberlandia (31%) and Araguari (20.6%). In total, 96.5% reside in urban areas.
Of the 29 reactive patients, one was hospitalized with severe symptoms (persistent vomiting, abdominal pain, fluid accumulation, and increase in hematocrit). This patient tested positive for R. rickettsii 18 days after symptom onset. Reported symptoms included fever, myalgia, rash, and vomiting, which are also common among other reactive cases (62% had fever/myalgia; 31%, vomiting; and 13.7%, rashes). Moreover, four other reactive cases involved rural or agricultural workers. These individuals engaged themselves in occupations related to potential exposure to Rickettsia sp.
Samples reactive at a dilution of 1:64, specifically for Rickettsia rickettsii and Rickettsia parkeri, represented 13.1% and 5.9% of all samples, respectively. Moreover, 20 samples were reactive to R. rickettsii, and nine to R. parkeri. Reactivity to R. rickettsii was higher among female patients. Of the 20 patients, 40% were aged from 18 to 40 years and 35% from 41 to 60 years. A total of 95% lived in urban areas. Table 1 shows their epidemiological profiles.
Sociodemographic characteristics of patients with clinical suspicion of dengue, whose samples were reactive in serological tests for the diagnosis of Rickettsia rickettsii and Rickettsia parkeri, Triangulo Mineiro, Minas Gerais State, Brazil, 2019
DISCUSSION
Spotted fever is endemic in Brazil and has expanded to rural, urban, and peri-urban areas, increasing exposure risk. Monitoring epidemiologiarel trends and identifying high-incidence regions is essential1. Early diagnosis is critical but challenging due to symptoms overlapping with other febrile illnesses and limited access to rapid testing11.
We found that 19% (29/152) of samples were reactive at a 1:64 dilution using IFA (the gold standard for the serological diagnosis of rickettsioses). Ideally, confirmation requires a fourfold or greater rise in IgG titters between paired serum samples collected at least 14 days apart12. However, due to the use of convenience sampling, only single samples were available, preventing definitive confirmation of spotted fever cases. Reactivity only at 1:64 dilution may suggest cross-reactivity with other Rickettsia species not primarily investigated. While R. rickettsii and R. parkeri are the main human pathogens in Brazil, other species, such as R. rhipicephali, R. amblyommatis, and R. bellii (the latter widely distributed and of uncertain pathogenicity), have also been reported13.
Previous serological surveys have highlighted cross-reactivity challenges in diagnosing spotted fever. Novo Cruzeiro, Minas Gerais State, showed a 10.1% seroprevalence for spotted fever agents, with 18% of sera showing IFA titers ≥1:64 for R. rickettsii, despite no symptoms in the preceding three years. The findings suggest a cross-reactivity that may have been caused by other Rickettsia species14. Another study in Piau, Minas Gerais, by Costa et al.15, reported frequent cross-reactivity among R. rickettsii, R. typhi, Coxiella burnetii, Bartonella spp., and Ehrlichia chaffeensis, likely due to shared antigenic determinants. The authors stressed the importance of simultaneously testing multiple rickettsial antigens to improve diagnostic accuracy. they noted no strong correlation between seropositivity and rural occupations. In contrast, our study found that four out of 29 reactive samples (13.7%) had come from individuals with outdoor professions, indicating a potential occupational risk.
Although most Rickettsia vectors, such as Amblyomma sculptum, A. aureolatum, and A. ovale, are typically associated with rural or peri-urban environments, most reactive cases in our study (96.5%) occurred in individuals residing in urban areas. Other Brazilian studies have found similar findings, suggesting that urban residents may still be exposed in visits to rural or natural areas for work or leisure or via peri-urban ecotones with vector-host interactions. Additionally, finding Rickettsia in urban cases may reflect increased mobility, underreporting of exposure history, or environmental changes that bring humans, reservoirs (e.g., capybaras, dogs), and vectors into closer contact. Note that Rhipicephalus sanguineus sensu lato, a species adapted to urban settings, has rarely been implicated as a vector of pathogenic Rickettsia species in Brazil3. These findings show the importance of considering spotted fever in patients from urban and non-endemic regions. Rickettsia sp. infections have been reported in dogs from non-endemic areas in southeastern Brazil, confirmed by serological and molecular analyses16. Given their outdoor habits and close human interaction, dogs serve as sentinels for SF transmission3.
The transmission of Rickettsia sp. to humans primarily depends on ticks, with Amblyomma sculptum and A. aureolatum configure as the main vectors in Brazil, and Rhipicephalus sanguineus considered a possible vector under specific conditions. However, current knowledge about tick vectors and their hosts, such as the capybara (H. hydrochaeris) remains limited, hindering a full understanding of the epidemiology of the disease. Accurate identification and mapping of tick species by region are essential to explain the transmission dynamics of rickettsioses. Moreover, the continuous discovery of new Rickettsia species and potential human infections highlights the complexity of their enzootic and epidemic cycles and the diversity of tick vectors. Rapid environmental changes driven by human activity further complicate these dynamics, reshaping host-vector interactions and potentially leading to new scenarios for tick-borne diseases17.
Human-driven environmental changes have increased interactions between humans and pathogens. In response, the One Health approach emphasizes collaboration between physicians, ecologists, and veterinarians, recognizing the interconnected health of humans, animals, and the environment. Sentinel animals such as horses and dogs have been used in spotted fever surveillance in endemic areas. Moreover, clinical suspicion should arise in any patient presenting symptoms such as fever, myalgia, headache, and rashes, especially in regions with high seroprevalence among sentinel species18.
Spotted fever presents symptoms such as fever, severe headache, nausea, vomiting, diarrhea, abdominal pain, muscle pain, limb paralysis, and gangrene, with red, non-itchy spots often appearing on wrists and ankles as the disease progresses. These manifestations resemble those of dengue, which include high fever, body aches, headache, rash, and warning signs such as abdominal pain, vomiting, and increased hematocrit10. In our study, one hospitalized patient showed such warning signs and was initially tested for dengue (non-reactive). However, serological analysis showed reactivity to R. parkeri (1:64), with reported symptoms of fever, myalgia, rash, and vomiting. In this study, of the 29 reactive samples, the most frequently reported symptoms included fever (62.07%), myalgia (62.07%), headache (58.62%), and back pain (44.83%), followed by gastrointestinal symptoms such as nausea (34.48%) and vomiting (31.03%). Although these signs are non-specific and overlap with other endemic febrile illnesses such as dengue, they are consistent with the clinical pattern of spotted fever in Brazil. According to Oliveira et al.19, fever, headache, myalgia, and prostration were the most prevalent symptoms among confirmed cases, reinforcing the relevance of these findings and the need for increased clinical suspicion in endemic or at-risk areas19. The results partially diverge from the epidemiological data in the literature, which indicates that rickettsiosis occurs more prevalently in adult men. However, of the 152 samples in this study, 85 (55.9%) came from women and 67 (44.1%) from men. Considering the greater representation of women in the sample, this may have influenced the higher proportion of reactivity in this group19.
Our findings showed rickettsial reactivity in patients initially suspected of having dengue, an endemic arbovirus in the region. This overlap may delay rickettsiosis diagnosis, highlighting the importance of evaluating patient history related to tick exposure. Given the high lethality of spotted fever, timely diagnosis is crucial. This study underscores the need to consider spotted fever in acute febrile cases, even at low antibody titers. The lack of paired samples and detailed patient exposure data limits deeper analysis, reinforcing the need for further epidemiological research in the region.
CONCLUSION
The findings showed rickettsial reactivity in suspected dengue patients, highlighting the need of considering spotted fever diagnosis even in non-endemic areas. Interdisciplinary collaboration can enhance epidemiological research and control strategies. Educational measures, including training healthcare providers, public awareness campaigns, and tick control, should be promoted.
DATA AVAILABILITY
The complete anonymized dataset supporting the findings of this study is included in this article.
ACKNOWLEDGMENTS
This study was partially funded by the Coordination for the Improvement of Higher Education Personnel – Brazil (CAPES) – Finance Code 001.
REFERENCES
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Editor:
Thelma Suely Okay https://orcid.org/0000-0001-9316-7288
