Rocky Mountain Spotted Fever, Panama

We describe a fatal pediatric case of Rocky Mountain spotted fever in Panama, the first, to our knowledge, since the 1950s. Diagnosis was established by immunohistochemistry, PCR, and isolation of Rickettsia rickettsii from postmortem tissues. Molecular typing demonstrated strong relatedness of the isolate to strains of R. rickettsii from Central and South America.


The Patient
On December 18, 2004, a 4-year-old female patient from a rural area west of the Panama Canal was admitted to Hospital del Niño, Panama City, Panama, with an 8-day history of intense headache, fever, malaise, myalgia, and arthralgia of the lower extremities and 3 days of generalized petechial rash. Ceftriaxone was administered empirically for suspected meningococcemia. Laboratory tests showed hyponatremia (126 mEq/L), hypoalbuminemia (2 g/dL), thrombocytopenia (48 × 10 9 cells/L), increased immature neutrophils (26%), and an elevated level of liver enzymes (aspartate aminotransferase 325 U/L and alanine aminotransferase 137 U/L). Results of routine blood cultures were negative. Cerebrospinal fl uid analysis showed only protein elevation. Serologic test results for equine encephalitis virus, dengue, hantavirus, and calicivirus as well as bacterial cultures were all negative. On December 18, the patient had seizures, which required intensive care management. Despite intense medical efforts, she died soon thereafter. The main autopsy fi ndings included myocarditis, interstitial nephritis, interstitial pneumonitis, encephalitis, and generalized lymphadenitis. Postmortem frozen unfi xed and formalin-fi xed tissues were sent to the US Centers for Disease Control and Prevention for etiologic assessment.
Immunohistochemical evaluation of formalin-fi xed, paraffi n-embedded tissues that used an immunoalkaline phosphatase technique demonstrated spotted fever group rickettsial antigens associated with rickettsia-like cells in vascular endothelium of multiple tissues, including heart ( Figure 1), lung, adrenal gland, and kidney. Serum collected on the day of death had immunoglobulin (Ig) G and IgM microimmunofl uoresence antibody titers of 2,048 to R. rickettsii antigen. DNA samples prepared from frozen brain, liver, lymph node, and spleen autopsy specimens were tested by PCR as described previously (7). When amplifi ed, a 208-bp fragment of the conserved 17-kDa Rickettsia antigen gene showed spotted fever rickettsial DNA was present in all autopsy tissues. An OmpA gene fragment (70-602 nt) was amplifi ed from brain and lymph node tissues (GenBank accession nos. DQ002503 and DQ002504). DNA sequencing of the ompA amplicons demonstrated that their nucleotide sequences were identical to each other and had 100% sequence similarity to the homologous ompA fragment of R. rickettsii strain Sheila Smith, isolated from a patient in Montana. An isolate of R. rickettsii (designated Panama 2004) was obtained from brain tissue in Vero E6 cells; its OmpA gene fragment (DQ164838) had 100% nucleotide sequence similarity to the reference sequence of R. rickettsii type strain Sheila Smith. R. rickettsii from Panama is similar to the other R. rickettsii strains circulating in Central and South America but differs from strain Sheila Smith in at least 1 locus containing tandemly repeated sequences (Figure 2).
The clinical characteristics of this patient, e.g., fever, headache, myalgia, petechial rash, and neurologic signs, initially aroused clinical suspicion for a viral fever caused by Venezuelan equine encephalitis (VEE) virus, particularly because there was a VEE outbreak in this area at that *Hospital del Niño, Panama City, Panama, and †Centers for Disease Control and Prevention, Atlanta, Georgia, USA time. The failure to diagnose a spotted fever rickettsiosis was partially the result of diminished awareness of RMSF among local physicians and absence of adequate specifi c diagnostic tests in Panama. Detection of hyponatremia, hypoalbuminemia, and thrombocytopenia, all observed in this patient, indicates endothelial vascular damage and increased vascular permeability and can aid in making a presumptive diagnosis (8). Death secondary to RMSF is associated with delays in diagnosis and delays in initiating appropriate antimicrobial therapy within the fi rst 5 days of the clinical disease (9). To prevent fatal outcomes, treatment of suspected case-patients should be initiated before the results of diagnostic tests are received, and treatment should be administered for 7-10 days or until the patient has been without fever for at least 3 days (9). R. rickettsii had been isolated previously in Panama from a few patients during the 1950s and from pools of A. cajennense ticks during the 1950s and 1960s (1,2,5). As occurred in this instance, cases of RMSF have likely been missed in Panama during the intervening decades; however, the low frequency of recognized cases of RMSF does not correspond to the relatively high seroprevalence of complement-fi xing antibodies to spotted fever group rickettsiae in residents of Panama (6). Indeed, inoculation of other pools from Amblyomma larvae and A. cajennense adults caused seroconversion to spotted fever group antigens in guinea pigs, but R. rickettsii was not isolated (5). Recent studies of Amblyomma ticks in Brazil have detected both R. bellii and an agent associated with A. americanum in the United States known as "Rickettsia amblyommii" (10). R. amblyommii has been implicated as a cause of a mild, self-limiting rickettsial illness in which seroconversion to R. rickettsii antigens occurs (11,12). R. parkeri, found in A. maculatum and A. triste, has been also identifi ed as a cause of disease in the United States and has been presumptively associated with infections of humans in Uruguay (13). Since the characterization of rickettsial agents associated with Amblyomma species that bite humans is a newly emergent fi eld in the Americas (13)(14)(15), other typhus group and spotted fever group agents may be found in Panama.

Conclusions
This case confi rms that R. rickettsii is still present in Panama. A high index of suspicion is necessary for an early diagnosis and empiric treatment of RMSF. Proposed widening of the Panama Canal and current construction of a new portion of the Trans-Istmus Highway disrupts the adjacent forest areas and would likely increase the frequency of human-tick contacts. Thus, RMSF should be considered  A tandem repeat region corresponding to 563048-563028 nt of the strain Sheila Smith genome and fl anking sequences were amplifi ed with AF (5′-GTGATTGCTATATTTCGCTTT-3′) and AR (5′-CTAAGATTTGTTCCGTATAGG-3′) primers as described elsewhere (7). Repeat sequence (GCCTTAT, indicated with brackets) present in 3 copies in strain Sheila Smith, whereas only 2 copies were present in R. rickettsii isolates from Brazil, Colombia, Costa Rica, and Panama. Homologous sequences of these strains are deposited to GenBank under the following accession nos.: DQ666020, R. rickettsii strain Panama 2004; DQ666021, R. rickettsii strain Brazil; DQ666022, R. rickettsii strain Colombia; DQ666023, and R. rickettsii strain Costa Rica. early in the differential diagnosis of febrile infections in tick-exposed persons in Panama. Further study of endemic rickettsioses, rickettsial agents, and possible tick vectors in Panama is warranted.
Dr Estripeaut is a pediatric infectious disease fellow at the Hospital del Niño, Panama. She specializes in tropical infectious diseases.