First Confirmed Human Case of Rickettsia parkeri Strain Atlantic Rainforest Infection on the North Coast of São Paulo State, Brazil

Simple Summary

Rickettsia parkeri strain Atlantic rainforest is an emerging pathogen in Brazil, although human cases are rarely reported. Here, we describe the first confirmed case in Caraguatatuba, São Paulo State. A 37-year-old man was bitten by an Amblyomma ovale tick in a forested area and subsequently developed fever, muscle pain, and a necrotic skin lesion at the bite site. Ticks collected from him and his dog were analyzed, and Rickettsia parkeri was successfully isolated from one specimen. The patient’s infection was confirmed by molecular testing. This represents the sixth documented occurrence in Brazil, highlighting the need for increased acarological surveillance in endemic regions where vector ticks are present.

Abstract

Rickettsia parkeri strain Atlantic rainforest is an emerging pathogen in Brazil, but human infections remain rarely reported. We report the first confirmed case in the municipality of Caraguatatuba on the northern coast of São Paulo State, Brazil. A 37-year-old man was bitten by an Amblyomma ovale tick while visiting a forested area. Six days later, he developed a papular skin lesion with a necrotic center at the tick bite site, accompanied by regional lymphadenopathy, myalgia, and flu-like symptoms. Ticks parasitizing both the patient and his dog were collected, along with the eschar. Two ticks were analyzed for Rickettsia isolation, and R. parkeri was successfully isolated from one infected specimen. The patient’s infection was confirmed by molecular testing through the PCR amplification of the gltA and ompA genes from an eschar inoculation sample. This represents the sixth confirmed case of rickettsiosis caused by R. parkeri in Brazil, and it reinforces the need for increased epidemiological surveillance in endemic regions for spotted fever caused by this pathogen. With the increasing recognition of R. parkeri in South America, further research is needed to better understand its transmission dynamics, clinical manifestations, and potential public health impact.

1. Introduction

Rickettsia parkeri is an obligate intracellular bacterium belonging to the spotted fever group Rickettsia (SFGR). It includes several strains that constitute a monophyletic lineage, such as Atlantic rainforest, NOD, and Parvitarsum, along with R. parkeri sensu stricto isolated from North and South America [1]. This pathogen is primarily transmitted by multiple Amblyomma tick species across the Americas [2].

Rickettsiosis caused by R. parkeri is the second most prevalent tick-borne disease attributed to Rickettsia of the spotted fever group (SFG) in the Americas, ranking second only to rickettsiosis caused by Rickettsia rickettsii [2,3]. In humans, signs and symptoms are nonspecific, similar to the prodromal phase of classical Rocky Mountain spotted fever, and these can resemble several other acute febrile infectious diseases, such as dengue, measles, and leptospirosis [4,5].

Although the symptoms are nonspecific, R. parkeri rickettsiosis presents local, regional, and systemic manifestations, mainly characterized by the inoculation eschar at the site of the tick bite, regional lymphadenopathy, fever, and rash, which are signs that aid in the diagnosis [6,7,8,9]. The inoculation eschar has been considered a characteristic clinical finding in R. parkeri rickettsiosis, and it also serves as an important source of DNA for the molecular detection and diagnosis of Rickettsia [8,10,11,12].

In Brazil, R. parkeri strain Atlantic rainforest (R. parkeri ARF) has been identified as the causative agent of tick-borne spotted fever, predominantly transmitted by Amblyomma ovale ticks [7,10,13]. Since 2007, approximately 1000 human cases of rickettsioses were confirmed in the State of São Paulo, Brazil. Among those, 44 have been reported in regions endemic for A. ovale ticks, 23 of which originated from the north coastal area of São Paulo State. These 23 cases are more likely to have been caused by R. parkeri ARF, but because of the lack of specific serological diagnosis, it is not possible to confirm the agent species.

In fact, despite the large number of cases, since the year 2009, in Brazil, only five clinical human cases of spotted fever caused by R. parkeri ARF were confirmed by DNA detection and sequencing [10,11,12,14,15]. While these five cases of R. parkeri infection have been documented in different regions of Brazil, this is the first confirmed human case of R. parkeri ARF in the north coastal region of São Paulo State, demonstrated by the DNA amplification and sequencing of the patient’s eschar sample, together with the epidemiological association and successful isolation of R. parkeri ARF from A. ovale ticks collected from the patient’s dog.

2. Materials and Methods

2.1. Case Presentation

The patient was a 37-year-old Brazilian man, resident of Caraguatatuba, a municipality on the north coast of São Paulo State, located at the foothills of Serra do Mar, covered by Atlantic rainforest biome.

He habitually visited forested regions, rivers, and waterfalls in the region, often accompanied by his dog, especially in the well-preserved riparian forest of the Guaxinduba River in the Cantagalo neighborhood. This area is also frequently visited by residents and tourists.

On 5 March 2023, he and his dog were parasitized by ticks while visiting Tronco Waterfall (S −23.58418/W −45.39314) (Figure 1A). At that time, he collected the tick specimens; however, in the following days, he did not develop any signs or symptoms.

On 23 July 2023, while hiking in the forest in Mancha Waterfall (S −23.59296/W −45.39770) (Figure 1B), he again noticed a tick attached to his right ankle, and he immediately removed and discarded it. Although he could not determine the exact attachment period, it is most likely that the period was between 6 to 12 h.

On 29 July (six days post-bite), the patient developed acute symptoms, including a cutaneous lesion with a surrounding macular eruption and a central necrotic ulcer at the bite site (Figure 2), as well as systemic symptoms such as inguinal lymphadenopathy, myalgia, and persistent headache. While the flu-like symptoms improved, the cutaneous lesion worsened, prompting him to seek medical attention on 31 July 2023, at a healthcare facility at Caraguatatuba. At this appointment, he was prescribed only anti-inflammatory medication.

On 2 August (four days after the onset of symptoms), the patient returned to the healthcare facility due to a lack of improvement of the cutaneous lesion. Non-specific laboratory tests were performed, including a complete blood count, aspartate aminotransferase (AST/TGO), alanine aminotransferase (ALT/TGP), gamma-glutamyl transferase (GGT), amylase, alkaline phosphatase, and creatinine quantification. The physician prescribed intravenous ceftriaxone for five days.

On 7 August (nine days after the onset of symptoms), the patient returned to the healthcare facility for follow-up testing, which included a complete blood count, and the quantification of urea, creatinine, and C-reactive protein (CRP). The physician prescribed oral ciprofloxacin for seven days, and no other medication was administered.

The patient reported full recovery from the disease symptoms a few days after 7 August, although the exact date is unknown. The inoculation eschar persisted for at least 58 days before completely healing, though the precise resolution date is unclear.

The patient claims his dog remained asymptomatic and required no treatment.

2.2. Molecular Investigation

After the onset of symptoms, the patient contacted our laboratory’s technical team. He voluntarily collected and submitted five tick specimens: four collected from his dog (date unknown) and one collected from him on 5 March 2023. The ticks were stored at room temperature in a plastic vial; two of the five ticks arrived live at the laboratory. Also, he sent to the lab a self-collected sample of the cutaneous eschar, which was obtained by manually removing the wound crust and storing it in a sterile microtube with 96% ethanol before shipping it to our laboratory for molecular analysis.

In the laboratory, ticks were morphologically identified using a stereomicroscope, as described by Barros-Battesti et al. [16]. DNA was extracted from the skin sample and tick specimens using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions.

The extracted DNA was then subjected to polymerase chain reaction (PCR) screening for Rickettsia spp. using the following two target genes: ompA (primers Rr190.70F and Rr190.602R) [17], htrA (primers 17k-5 and 17K-3) [18], and gltA (primers CS-78 and CS-323) [19]. PCR amplification was carried out using DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and using the cycling conditions as previously described in the literature. Positive (R. parkeri) and negative (autoclaved DNA-free Milli-Q water) controls were included in each reaction.

PCR products were analyzed by electrophoresis on a 2% agarose gel. Amplicons of the expected size were purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently sequenced using the same primers applied in the amplification step with BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3500 Genetic Analyzer (both from Applied Biosystems, Foster City, CA, USA). The obtained sequences were manually edited to generate consensus sequences using ChromasPro 1.5 (Technelysium, Qld, Australia) and aligned using the ClustalW algorithm in MEGA X v10.1.7 [20].

Consensus sequences were compared with those available in the GenBank database using the online Basic Local Alignment Search Tool (NCBI BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 May 2025) to assess consensus similarity. Gene fragments were concatenated and subjected to phylogenetic analysis to determine their relationship with known Rickettsia species.

A phylogenetic tree was constructed using the maximum likelihood method in PHYML (v3.0), employing the General Time Reversible (GTR) + G evolutionary model [21]. Bootstrap support values were calculated based on 1000 replicates, and the nodes were supported by high bootstrap values, confirming the taxonomic identity of the pathogen. All sequences generated in this study have been deposited in GenBank under accession numbers PV890545-53.

2.3. Isolation of Rickettsia from Live Ticks

Two live A. ovale adult ticks collected from the dog were subjected to Rickettsia cell culture isolation by the shell vial technique, as described [18]. Briefly, shell vials seeded with Vero cells were inoculated with tick-body homogenates, centrifuged (700× g for 60 min), and incubated at 28 °C. The percentage of Vero cells infected with rickettsiae was monitored by Giménez staining of cells scraped from each inoculated monolayer. The rickettsial isolate was considered established in the laboratory after at least 3 Vero cell passages, each exceeding 90% of infected cells. DNA was extracted from the 3^rd passage-infected cells and subjected to PCR targeting gltA, htrA, and ompA genes to confirm R. parkeri infection (as described above). Remnants of the two ticks used to inoculate shell vials were submitted to DNA extraction, as done with the other ticks.

3. Results

The results of the hemogram and blood biochemistry performed on 2 August presented the following altered laboratory parameters: discrete lymphocytopenia [804/mm³ (reference values: 1000–3300/mm³)], discrete elevation of ALT [43 U/L (reference values: 10–40 U/L)], and elevated alkaline phosphatase [316 U/L (reference values: 20–150 U/L)] and creatinine [1.47 mg/dL (reference values: 0.40–1.30 mg/dL)].

The hematologic and biochemical test performed on 7 August presented discrete monocytosis [808/mm³ (reference values: 150–800/mm³)], lymphocytosis [7070/mm³ (reference values: 3500–6500/mm³)], and an increased CRP [12 mg/L (reference values: less than 6 mg/L)].

All five ticks were identified as A. ovale adults, one from the patient and four from the dog. Among the two ticks processed by the shell vial technique, rickettsiae were successfully isolated from an A. ovale female. This isolate was established in Vero cells for several passages; frozen stocks have been deposited at the rickettsial collection of our laboratory and designed isolate Caragua have been deposited in Genbank under accession numbers PV890551, PV89052, and PV890553.

Molecular analyses were performed on all five ticks, the inoculation eschar, and the Vero cell rickettsial isolate. Part of the samples yielded the successful amplification of partial sequences of the gltA, htrA, and/or ompA genes (

Table 1. Detection of gltA, htrA, and ompA genes in different sample types (eschar, tick, and isolate). Check marks (X) indicate successful amplification of partial gene sequences by PCR.

Sample	Sample of Type	gltA	htrA	ompA
E01	Eschar	X		X
T02	Tick			X
T04	Tick 1	X	X	X
IS01	Isolate	X	X	X

¹ This tick is the same from which the rickettsial isolate was obtained.

).

After DNA sequencing the PCR amplicons, BLAST analysis revealed that the sequences of gltA (350 bp) and htrA (497 bp) were 100% identical to the reference genome of R. parkeri ARF (GenBank accession no. CP040325). For the ompA gene, the sequence obtained from the sample of the inoculation eschar was 100% identical (346 bp) to R. parkeri ARF (CP040325), although only a 346 bp fragment could be obtained with certainty due to non-reliable reads in the two flanks of the chromatogram. The ompA sequencing of samples T02, T04, and IS01 showed they were 100% (491 bp) identical to R. parkeri ARF (CP040325). The gltA, htrA, and ompA sequences generated in this study have been deposited in GenBank under accession numbers PV890545-53.

Phylogenetic analyses were conducted using two concatenated datasets, comparing our sequences with those available in GenBank. The first dataset included 32 sequences with a total length of 844 nucleotides (including gaps) generated from the genes gltA and ompA. The second dataset included 30 sequences with 1343 sites in total (including gaps) from the genes gltA, htrA, and ompA. In both analyses, samples from Caraguatatuba were found to be identical to R. parkeri ARF (Figure 3 and Figure 4).

Sequences derived from the inoculation eschar, two A. ovale ticks, and the bacterial isolate were shown to be 100% identical to the reference sequences of this strain. The phylogenetic tree based on partial sequences of the gltA and ompA genes demonstrated that all sequences obtained in this study clustered within the R. parkeri strain Atlantic rainforest clade (Figure 3). A second phylogenetic analysis was conducted using concatenated sequences of the gltA, htrA, and ompA genes, and this included only the bacterial isolate and the A. ovale tick from which the isolate was obtained (Figure 4).

The resulting topologies demonstrate that the sequences obtained in this study consistently cluster with the reference sequences from GenBank, confirming the identification of R. parkeri ARF. In addition, the tree based on the three-gene dataset (Figure 3) provides a clearer demonstration of the genetic relationships between R. parkeri and other closely related Rickettsia species. This analysis further confirmed the placement of these sequences within the R. parkeri ARF, with high bootstrap support. The genetic congruence between the tick and the isolate reinforces the accuracy of the isolation and identification procedures.

4. Discussion

The present case represents the sixth confirmed occurrence of rickettsiosis caused by R. parkeri in Brazil, exhibiting a clinical pattern consistent with previous reports [10,11,12,14,15]. The incubation period for R. parkeri infection typically ranges from 4 to 10 days, and in this case, symptoms developed six days after the tick parasitism, aligning with previously documented cases [12]. The successful detection of R. parkeri DNA in tick specimens collected from the dog further supports that the A. ovale tick is epidemiologically associated with this pathogen in the Atlantic rainforest biome.

The presence of A. ovale in the Atlantic rainforest biome is well-documented, with previous studies identifying this tick species as the primary vector of R. parkeri in Brazil [12,13]. The growing number of cases of spotted fever in São Paulo State, particularly in regions where A. ovale is endemic, highlights the need for continued epidemiological surveillance. Between 2007 and 2023, 44 cases of spotted fever were confirmed in areas with A. ovale prevalence in São Paulo State, 23 of which originated from the municipality of Caraguatatuba [22].

The municipality has a population of approximately 135,000 inhabitants [23] and experiences a high influx of tourists on weekends, with the population doubling during holidays. According to the Caraguatatuba Municipal Government [24], during the peak summer season of 2024/2025, for instance, an estimated 1.6 million visitors were recorded. These findings reinforce the importance of this region as an area of significant public health concern.

Confirmed human cases of rickettsiosis caused by R. parkeri in Brazil may be underreported, largely due to their clinical similarities with other febrile illnesses such as dengue, leptospirosis, and other rickettsioses, including R. rickettsii. The mild to moderate clinical presentation of rickettsiosis caused by R. parkeri, characterized by fever, headache, myalgia, regional lymphadenopathy, and the presence of an inoculation eschar, further complicates clinical diagnosis [6]. This case highlights the need for increased clinical awareness and molecular diagnostic approaches to improve the identification of R. parkeri infections.

In the present case, the patient had a history of frequent exposure to forested environments and close contact with domestic animals, factors that increase the risk of tick exposure and subsequent infection. The role of domestic animals, particularly dogs, as a primary host or mechanical carriers of A. ovale has been previously suggested [13,25]. In this context, pet owners who frequent endemic areas should be aware of tick prevention measures, including the regular use of acaricides and routine tick checks on both humans and animals after outdoor activities.

From an ecological perspective, the maintenance of R. parkeri in enzootic cycles involves a complex interplay between tick vectors, wildlife amplifiers, and environmental factors. Studies have identified small rodents as potential amplifiers of R. parkeri [26].

In particular, the Atlantic Forest biome, characterized by high biodiversity and extensive human–wildlife interaction, serves as an ideal setting for the transmission of tick-borne pathogens. The encroachment of human populations into these preserved areas, combined with ecological changes and deforestation, may contribute to the increasing incidence of rickettsial infections.

The analysis of laboratory tests performed on 2 August revealed alterations compatible with an immune response and possible hepatic and renal impairment. The discrete lymphocytopenia (804/mm³) may indicate an initial inflammatory response or transient immunosuppression. The elevation of ALT (43 U/L) and alkaline phosphatase (316 U/L) suggest hepatic involvement, possibly associated with an ongoing infection. Additionally, the increase in creatinine (1.47 mg/dL) may indicate mild renal dysfunction, possibly resulting from the infection or a systemic inflammatory state.

In the second test, conducted on 7 August, an inversion of the cellular pattern was observed with discrete monocytosis (808/mm³) and an increased number of neutrophils (7070/mm³), reinforcing the presence of an evolving inflammatory response. Furthermore, the elevation of CRP (12 mg/L) confirms the presence of active inflammation, which may be related to an ongoing infectious process.

These findings are consistent with previous studies on R. parkeri infections, where altered laboratory parameters were commonly observed. Among the confirmed cases reported in the literature [9], 63% presented laboratory abnormalities, with 50% showing elevated AST, 42% leukopenia, and 42% increased ALT. Additionally, some cases exhibited elevated alkaline phosphatase, thrombocytopenia, increased lactate dehydrogenase, and other hematologic abnormalities [6,27,28].

Similarly, probable cases demonstrated a high prevalence of leukopenia (50%), with some also exhibiting increased CRP levels [29]. The laboratory findings in our study align with these previously reported alterations, further supporting the diagnosis of R. parkeri infection.

The molecular analysis of the collected samples revealed the presence of the R. parkeri ARF strain in two of the collected ticks and in the inoculation eschar sample. The partial amplification of the gltA, htrA, and/or ompA genes and their subsequent BLAST analysis confirmed their identity with sequences deposited in GenBank, demonstrating a strong relationship with the Atlantic rainforest strain (CP040325). The phylogenetic analysis of the concatenated sequence showed that the samples from Caraguatatuba were identical to this strain.

Although serology is considered the standard approach for the diagnosis of spotted fever, no blood sample was collected for serological testing because the disease was not initially suspected by the medical team. Despite this, the molecular findings, including the detection and sequencing of Rickettsia parkeri DNA from patient samples, provide robust evidence to support the diagnosis. These results are consistent with previously reported cases and highlight the importance of molecular methods in confirming infections when the disease is not clinically recognized at the time of presentation.

Rickettsiae are intracellular bacteria insensitive to beta-lactam drugs, such as ceftriaxone, but it is known that ciprofloxacin presents some anti-Rickettsia activity, and fluoroquinolones are generally not considered effective against rickettsial infections [30] because they are about ten times less effective than specific antibiotics such as doxycycline, which is considered the first-line treatment [31]. Nevertheless, the patient claimed to have achieved full recovery a few days (not precise) after ciprofloxacin treatment.

The treatment regimen prescribed in this case deviated from the standard recommendation for rickettsial infections, which typically involves doxycycline as the first-line antibiotic [4]. The initial use of ceftriaxone followed by ciprofloxacin raises concerns about the potential mismanagement of R. parkeri rickettsiosis, as fluoroquinolones are generally not considered effective for rickettsial infections [30]. This highlights the need for enhanced medical training to ensure that clinicians in endemic areas recognize and appropriately manage suspected rickettsial diseases. Finally, strengthening surveillance in public health efforts by integrating molecular diagnostics, tick monitoring programs, and seroepidemiological studies would provide a more accurate understanding of the distribution and prevalence of R. parkeri in Brazil.

Future research should also focus on identifying additional vector species, amplifier hosts, and potential environmental factors that may influence the occurrence and spread of this pathogen. By improving diagnostic capabilities and public health preparedness, the burden of rickettsial diseases in Brazil can be mitigated.

5. Conclusions

We report the first case of R. parkeri ARF infection in the northern coast of São Paulo State, Brazil. Molecular confirmation was achieved through the PCR amplification of ompA and gltA genes from an inoculation eschar sample. This case reinforces the importance of continuous epidemiological monitoring, public health awareness, and appropriate tick-bite prevention measures in endemic regions. Further studies are required to better understand the ecological role of the A. ovale tick and its potential role as a vector of R. parkeri in Brazil.