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.


Isolation of Rickettsia rickettsii in Rocky Mountain Spotted Fever Outbreak, Panama
Yamitzel Zaldívar, Michelle Hernández, Lillian Domínguez, Lisseth Saénz, Santiago Montilla, Maria E. Barnett de Antinori, Felipe S. Krawczak R ocky 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 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.
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 follow- The diagnosis of severe cases of RMSF in Piedra Roja represents a new locality for this disease in Pan-ama. 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.