Volume 27, Number 4—April 2021
Isolation of Rickettsia rickettsii in Rocky Mountain Spotted Fever Outbreak, Panama
We report new cases of Rocky Mountain spotted fever in patients from Kinkantu, Ngäbe-Bugle indigenous comarca, Panama. We isolated Rickettsia rickettsii in cell culture after intraperitoneal inoculation of guinea pigs with tissues from a deceased patient. Our results indicate that Rocky Mountain spotted fever is emerging in this region.
Rocky Mountain spotted fever (RMSF) causes severe cases of rickettsiosis and is considered a principal tickborne pathogen in the Americas (1). Clinical suspicion is crucial for timely therapy with doxycycline to prevent severe illness and death (1). In Panama, 5 cases of RMSF were reported during 1950–1953, of which 2 were fatal; since 2004, a total of 19 new cases have been reported in Panama, with 13 fatal cases (2). We report new cases of RMSF from Piedra Roja, a rural village of Kankintu, Ngäbe-Bugle indigenous comarca, located at 750 m above sea level in the western mountainous region of Panama without road access.
In February 2019, a total of 7 persons 3–20 years of age from a family cluster had a clinical picture characterized by temperatures of 39°C–41°C (100%), generalized exanthema (100%), diarrhea and vomiting (86%), headaches (71%), severe dehydration (57%), abdominal pain (43%), and hepatomegaly and jaundice (29%). The patients reported no history of recent tick bites or attachment; according to each patient, the duration of symptoms varied from 9 to 11 days. Of these 7 patients, 2 recovered after treatment with doxycycline, 1 recovered without treatment with doxycycline, and 4 died.
We diagnosed rickettsiosis by PCR on blood and samples of spleen, liver, brain and lung, using the Rr190.70p and Rr190.602n primers, which amplify a ≈532 bp fragment of outer membrane protein gene (ompA) (3). Samples of blood, liver, and spleen from 6 patients yielded ompA amplicons, of which 3 generated DNA sequences 100% identical to R. rickettsii were deposited in GenBank (accession nos. MF678551.1, KX363464.1, and CP006010.1).
Tissue samples were recovered during the autopsy of 1 patient and stored at −40°C. Because this temperature is higher than that recommended to keep Rickettsia viable, we inoculated 1 guinea pig (Cavia porcellus) with tissue homogenate to avoid rickettsial load loss at the moment of isolation. These animals have been reported as amplifier hosts for R. rickettsii (4,5). Therefore, we inoculated a homogenate of spleen, liver, and lung tissues into an adult male guinea pig before starting the isolation through cell culture. The animal did not have a fever (rectal temperature ≤39.6°C) but died on the 7th day postinoculation (dpi). We extracted and macerated the liver, spleen, brain, and lungs to inoculate 5 additional guinea pigs (second passage), following Krawczak et al. (4). Of these, 2 animals died <24 hours later and were eliminated from the study, 1 developed high fever (>40.0°C) at 4 dpi that persisted until 6 dpi, and 2 remained afebrile but died at 4–5 dpi. We isolated rickettsiae in cell culture from a febrile (>39.6°C) guinea pig that was euthanized at 6 dpi. We inoculated fragments of liver, spleen, and lungs into flasks containing a monolayer of Vero cells, as previously described (5,6). We considered a rickettsial isolate to be established in the laboratory after third passages, each reaching an infected cell level >90% (6,7). We successfully isolated rickettsiae in Vero cells of homogenate derived from a 3-guinea-pig passage.
We extracted DNA from infected cells following Krawczak et al. (4) using a PCR targeting gltA (401 bp), ompA (532 bp), and ompB (511 bp) (3,6). Sequenced PCR products showed a 100% identity with R. rickettsii gltA (GenBank accession nos. CP018914.1, CP018913.1, CP006010.1, CP006009.1, and CP000766.3), ompA (GenBank accession nos. MF678551.1 and MF988095.1), and ompB (GenBank accession nos. CP018914.1, CP018913.1, CP006010.1, CP006009.1, and CP000766.3). We deposited DNA of an isolate in GenBank (accession no. MT814706 for the gltA gene, MT268770 for the ompA gene, and MT814707 for the ompB gene). We designated the R. rickettsii isolate as strain NB, for Ngäbe Bugle, and deposited it in the Gorgas Memorial Institute at Biosafety Level 3.
The diagnosis of severe cases of RMSF in Piedra Roja represents a new locality for this disease in Panama. RMSF has been reported previously from the provinces of Panama, Panama Oeste, and Colon, associated with the distribution of Amblyomma mixtum and Rhipicephalus sanguineus s.l. ticks (2,8). More studies will be needed to determine the ecology related to these cases.
We were able to isolate R. rickettsii from infected tissues stored at −40°C, which is higher than the recommended temperature of −80°C for preserving tissues (9). Because of the relevance of R. rickettsii as a pathogen, the isolation of strains favors obtaining antigens for serologic tests and for further studies to determine the genetic and pathogenic differences between strains. Currently, >30 genotypes of R. rickettsii exist, with different degrees of pathogenicity; therefore, a more representative sample of isolates may make it possible to estimate variations among different populations (10).
In summary, we investigated an outbreak of RMSF in Piedra Roja, a rural village in western Panama, an area where this disease had not previously been reported. Clinicians should remain aware of the possibility of R. rickettsii infection in this region.
Ms. Zaldívar is a medical technologist and head of the Department of Research-Surveillance and Biological Risk 3, Gorgas Memorial Institute for Health Research, Panama City, Panama. Her primary research interest is molecular biology and genetics.
We thank Hermes Santos, Doristela Isaza, Brechla Moreno, and Claudia Gonzalez for their assistance; Sebastián Muñóz-Leal for his contributions to sequencing; Joao Varela-Petrucelli for his comments; Hector Cedeño for the information provided on the outbreak; and Lance Durden for corrections in English.
This work was authorized by the Gorgas Memorial Institute’s Bioethics Committee of Investigation (no. 582/CBI/ICGES/19) and Institutional Animal Care and Use Committee (no. 01-2020), and financed with US HHS-ASPR Cooperative Agreement funds (IDSEP 170040) coordinated by Maria E. Barnett de Antinori.
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Original Publication Date: March 10, 2021