Clinical Specimens

Clinical specimens for confirmatory testing of suspected rickettsial diseases were submitted to the California Department of Public Health Viral and Rickettsial Disease Laboratory (CDPH-VRDL) for public health surveillance purposes and considered exempt from human subject regulations by the California Health and Human Services Agency Committee for the Protection of Human Subjects (project no. 2023-085). In addition to a plasma specimen from the initial case-patient, archival clinical specimens (serum or plasma) collected over the past 20 years from 8 confirmed SFG rickettsiosis case-patients were available for molecular characterization. SFG rickettsiosis cases were classified as confirmed for public health surveillance purposes on the basis of clinical and laboratory criteria (12).

Nucleic Acid Extraction and Real-time PCR

We extracted total nucleic acids using the NucliSENS easyMAG instrument (bioMérieux, https://www.biomerieux.com) with a serum or plasma input volume of 300 µL and a total nucleic acids output volume of 110 µL. We further concentrated total nucleic acids from case-patient 2 by 4-fold using the RNA Clean and Concentrator Kit (Zymo Research, https://www.zymoresearch.com).

We tested specimens for Rickettsia using a laboratory-developed triplex real-time reverse transcription PCR (rRT-PCR) targeting a R. rickettsii–specific 23S rRNA single-nucleotide polymorphism (SNP), a R. typhi–specific 23S rRNA SNP, and genus-specific regions of the 23S rRNA (Appendix). The assay was developed and the performance specifications established by the CDPH-VRDL as required under the Clinical Laboratory Improvement Amendments. A R. rickettsii–specific real-time PCR (rPCR) assay, RRi6, was performed as described by Kato et al. (13); the assay does not detect Rickettsia 364D (14).

MLST

We amplified and sequenced 6 target sequences, 23S rRNA, 16S rRNA, gltA, ompA, ompB, and sca4 (Appendix); the last 5 of those targets correspond to the regions recommended for the gene sequence–based classification of Rickettsia (15). A commercial laboratory performed bidirectional Sanger sequencing (ELIM Biopharmaceuticals, https://elimbio.com). We performed sequence editing and assembly using Geneious Prime 2022.0.2 (https://www.geneious.com). Nucleotide BLAST searches of the National Center for Biotechnology Information (NCBI) nucleotide collection and whole-genome shotgun contigs databases (https://blast.ncbi.nlm.nih.gov) were performed to identify genetic near neighbors and facilitate phylogenetic analyses. We concatenated and aligned the 16S rRNA, gltA, ompA, ompB, and sca4 sequences for validly named SFG Rickettsia species and constructed a maximum-likelihood phylogenetic tree using MEGA 10 (https://www.megasoftware.net). We measured the reliability of the resulting tree by bootstrap resampling using 1,000 replicates.

Tick Collection and Testing

We conducted field investigations at potential exposure sites by flagging vegetation and leaf litter for ticks. We identified tick species, sex, and life stages by examining morphologic features. For the 2023 case investigation, we processed adult ticks collected from Alameda and Contra Costa Counties for nucleic acid extraction using the DNeasy Blood and Tissue Kit (QIAGEN, https://www.qiagen.com). We screened tick nucleic acid extracts for R. rickettsii using the RRi6 assay. Ticks collected and identified from San Mateo and Marin Counties during the 2004 case investigation were screened for R. rickettsii DNA by the US Army Center for Health Promotion and Preventative Medicine–West.

Design and Development of a Rickettsia sp. CA6269 Real-Time PCR

We performed a nucleotide BLAST search using the ompA sequence from Rickettsia sp. CA6269 (GenBank accession no. JN990595) and downloaded and aligned selected sequences using the multiple sequence alignment tool. We selected regions of sequence divergence for primer and probe design using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast). We designed a 5′ exonuclease rPCR to amplify and detect a 146 bp region of ompA. The assay oligonucleotides were synthesized by Integrated DNA Technologies (https://www.idtdna.com). The 25-µL reaction mixture consisted of RLompAF1 (5′-GGGCACTTGGTGTTCCTACA-3′) and RLompAR1 (5′-AAATGCCCAATTGTTTTGAGGAC-3′) primers at 500 nM, RLompAP1 (6FAM- CTAATGGTG/ZEN/ATCCTACTGGCG-3IABkFQ) probe at 100 nM, 1X Premix ExTaq (Probe qPCR) mix (Takara Biosciences, https://www.takarabio.com), and 5 µL of total nucleic acids. We performed amplification and fluorescence detection with the ABI 7500 FAST DX Sequence Detection System (ThermoFisher Scientific, https://www.thermofisher.com) using the fast mode and the following amplification conditions: 1 minute at 95°C, 45 cycles of 95°C for 3 seconds, and 60°C for 30 seconds. We assessed assay analytical specificity by using a panel of 37 total nucleic acid extracts from members of the order Rickettsiales (Appendix) and evaluated analytical sensitivity by using 10-fold serial dilutions of a quantified 156-bp gBlock gene fragment (Integrated DNA Technologies) spiked into nucleic acids from pooled human blood and tested in replicates of five. We defined the assay limit of detection as the smallest number of DNA copies detected per reaction for all 5 replicates.

Case-Patient 1

The CDPH was notified of a suspected RMSF case involving a man with symptom onset in July 2023. The case-patient sought care at the emergency department (ED) with a 3-day history of influenza-like symptoms; he experienced fever, headaches, myalgias, arthralgias, malaise, loss of appetite, nonbloody diarrhea, and left-sided abdominal pain. Vital signs included temperature of 103°F (39.4°C), pulse of 96 beats/min, blood pressure of 116/71 mm Hg, and respiratory rate of 16 breaths/min. Lower left quadrant and suprapubic abdominal tenderness were noted upon physical examination. Of note, a rash was not observed. An antimicrobial regimen of ceftriaxone, metronidazole, and oral vancomycin was initiated, and the case-patient was hospitalized. The case-patient was transferred to the intensive care unit (ICU) on day 3 of hospitalization because of worsening hypoxemia, acidosis, encephalopathy, and seizures. Doxycycline was added to the treatment regimen on day 3 of hospitalization after an infectious diseases consultation that included rickettsial diseases in the differential diagnosis because of the severity of illness and clinical manifestations (Table 1).

Serologic testing for SFG Rickettsia by a commercial laboratory failed to detect a significant IgG titer in an acute serum specimen collected 5 days after symptom onset (Table 1). All blood cultures were negative. A laboratory diagnosis of rickettsial infection was initially made with the Karius Test, a microbial cell–free DNA (mcfDNA) metagenomic sequencing method (16). Sequencing of a plasma specimen collected 7 days after the onset of symptoms determined that R. rickettsii detected at 254,523 DNA molecules/microliter and R. slovaca detected at 97,653 DNA molecules/microliter were the best matches in the Karius Test genomic reference database. Serologic testing of a convalescent serum specimen, collected 105 days after symptom onset, demonstrated a significant IgG titer of >1:256 for SFG Rickettsia. The case-patient was discharged after being hospitalized for 22 days, including 11 days in the ICU, with a primary diagnosis of RMSF and secondary diagnoses of septic shock, acute kidney injury caused by acute tubular necrosis requiring intermittent hemodialysis, acute hypoxemic respiratory failure, encephalopathy, seizures, diarrhea, hyponatremia, abnormal liver enzymes, thrombocytopenia, supraventricular tachycardia, atrial fibrillation, and gangrene involving multiple digits on both hands.

Figure

Figure. Maximum-likelihood phylogenetic tree of concatenated multilocus sequences in study of newly recognized spotted fever group Rickettsia as cause of severe Rocky Mountain spotted fever–like illness, northern California, USA. ...

The residual plasma specimen from mcfDNA sequencing was provided to the CDPH-VRDL for species-specific confirmatory testing. We tested total nucleic acids extracted from this sample with the triplex rRT-PCR that detects a Rickettsia-specific sequence, an R. rickettsii–specific SNP, and an R. typhi–specific SNP in the 23S rRNA. Unexpectedly, only the Rickettsia species target, and not the R. rickettsii–specific SNP, was detected (cycle threshold [Ct] value 24.9) (Table 2). Subsequent testing with the RRi6 assay detected DNA at a Ct value of 30.5 (Table 2). To address the discordant R. rickettsii test results for the triplex rRT-PCR and the RRi6 assay, we sequenced a 1,468-nt segment of the rickettsial 23S rRNA (GenBank accession no. OR600926). Comparative sequence analysis revealed a 2-nt difference from Rickettsia 364D and a 3- to 4-nt difference from R. rickettsii, including an alternate SNP allele for the rRT-PCR SNP target. We further assessed species relatedness using a MLST scheme established for the classification of Rickettsia (15). We amplified and sequenced a 1,408 nt segment of 16S rRNA, a 1,051 nt segment of gltA, a 587 nt segment of ompA, a 4,849 nt segment of ompB, and a 2,944 nt segment of sca4 (GenBank accession nos. OR600927 and OR596747–50). Searches of the NCBI nucleotide databases revealed perfect sequence matches with gltA (290 nt) and sca4 (673 nt), a single nucleotide insertion/deletion difference in ompA (471 nt), and a single SNP difference in ompB (758 nt) from Rickettsia sp. CA6269 (GenBank accession nos. JN990594–7) (11). MLST results indicated that the strain, designated CA23RL1, was nearly identical to Rickettsia sp. CA6269. In contrast, CA23RL1 displayed significant sequence divergence from R. rickettsii and Rickettsia 364D at each MLST target (Table 3). Phylogenetic analysis of concatenated sequences from validly named SFG Rickettsia demonstrated that CA23RL1 occupied a distinct branch and was most closely related to R. rickettsii (Figure).

The case-patient had not traveled outside of the San Francisco Bay area within 19 days of illness onset and did not recall a tick bite. The case-patient had played golf at 5 courses located in Alameda and Contra Costa Counties within 14 days of symptom onset, including the day symptoms began. Of note, the case-patient recalled entering vegetation to retrieve golf balls at several courses. Because that activity might have been a potential source of exposure to ticks, as follow-up we collected ticks from the 5 golf courses within 16–22 days (late July and early August) of illness onset. In all, 197 adult ticks were collected and identified as either D. occidentalis (n = 135) or D. similis (n = 62). None of the ticks were infected with R. rickettsii or Rickettsia sp. CA6269 as determined by the RRi6 assay.

Case-Patient 2

We identified case-patient 2 through retrospective testing of 8 confirmed SFG rickettsiosis cases with the triplex rRT-PCR and RRi6 assays (Table 2). We detected Rickettsia spp. (Ct value 34.3), but not R. rickettsii or R. typhi, using the triplex rRT-PCR. The RRi6 assay detected rickettsial DNA (Ct value 34.3). Amplification and sequencing of a 397-nt segment of 23S rRNA (GenBank accession no. OR600925) by nested RT-PCR revealed an exact match to the 23S rRNA sequence determined for case-patient 1 and a 2- to 4-nt difference from Rickettsia 364D and R. rickettsii, supporting the identification of a second case of Rickettsia sp. CA6269 infection.

Case-patient 2, a male adult, was diagnosed with RMSF in 2004 on the basis of clinical features and a 4-fold increase in serologic titer to SFG Rickettsia in serum specimens collected 10 days apart (Table 1). Illness onset occurred in June; the case-patient sought care at the ED with a 5-day history of headaches, vomiting, photophobia, neck pain, and confusion. Upon initial examination, vital signs included a body temperature of 101.8°F (38.8°C), pulse of 96 beats/min, blood pressure of 143/89 mm Hg, and respiratory rate of 20 breaths/min. Physical examination revealed mild nuchal rigidity, a maculopapular rash on the arms and legs, and leg edema. The case-patient was hospitalized on the basis of the ED assessment that included encephalitis, sepsis, hypoxemia, rash of unknown etiology, and chronic leg edema, and antimicrobial treatment with ceftriaxone, vancomycin, and acyclovir was initiated. On day 3 of hospitalization, the patient became hypoxic and comatose and was intubated and transferred to the ICU for 4 days. Doxycycline was added to the treatment regimen on day 3 of hospitalization after an infectious diseases consultation that included rickettsioses in the differential diagnosis on the basis of clinical manifestations and the patient’s outdoor activities. Results of blood, CSF, urine, and sputum cultures were negative for significant pathogens. Serologic test results on day 10 of hospitalization indicated an IgG titer of 1:4,096 for SFG Rickettsia. After 13 days, the patient was discharged with a primary diagnosis of Rickettsia encephalitis.

The case-patient had not traveled outside the San Francisco Bay area during the 2 weeks before onset but had visited and camped at a county park and state beach in San Mateo County and a rural community in Marin County within that period. At the county park, he recalled finding a tick crawling on his body but not a tick bite. Field investigations at these locations conducted 30–40 days (July) after illness onset yielded 10 D. occidentalis ticks, 8 D. similis ticks, and 6 Ixodes pacificus ticks. Molecular testing of the Dermacentor spp. ticks did not detect R. rickettsii.

Rickettsia sp. CA6269–Specific Real-time PCR

After the sequence-based identification of Rickettsia sp. CA6269 infections, we developed an rPCR targeting genotype-specific regions of ompA. We did not observe assay cross-reactivity with nucleic acids from Anaplasma phagocytophilum, Ehrlichia chaffeensis, Orientia tsutsugamushi, and 14 Rickettsia species, including 10 R. rickettsii strains and 11 Rickettsia 364D strains (Appendix). The assay limit of detection was 1 copy of DNA per reaction. Of 9 SFG rickettsiosis case-patients tested, Rickettsia sp. CA6269 was detected only for case-patient 1 (Ct value 29.6) and case-patient 2 (Ct value 34.4) (Table 2). The remaining 7 case-patients were confirmed R. rickettsii infections on the basis of detection of the species-specific 23S rRNA SNP with the triplex rRT-PCR.