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Investigation of an outbreak of rickettsial febrile illness in Guatemala, 2007

  • Author Footnotes
    1 Jiann-Ping Hsu College of Public Health, Georgia Southern University, 501 Forest Dr., Statesboro, GA 30458, USA.
    Marina E. Eremeeva
    Footnotes
    1 Jiann-Ping Hsu College of Public Health, Georgia Southern University, 501 Forest Dr., Statesboro, GA 30458, USA.
    Affiliations
    Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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  • Elsa Berganza
    Affiliations
    Área de Salud, Jutiapa, Guatemala

    Field Epidemiology Training Program, CDC Regional Office for Central America and Panama, Guatemala City, Guatemala
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  • Gloria Suarez
    Affiliations
    Centro Nacional de Epidemiología, Ministerio de Salud Pública y Asistencia Social, Guatemala City, Guatemala
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  • Lorena Gobern
    Affiliations
    Laboratorio Nacional de Salud de Guatemala, Ministerio de Salud Pública y Asistencia Social, Guatemala City, Guatemala
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  • Erica Dueger
    Affiliations
    Centro Nacional de Epidemiología, Ministerio de Salud Pública y Asistencia Social, Guatemala City, Guatemala
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  • Leticia Castillo
    Affiliations
    Laboratorio Nacional de Salud de Guatemala, Ministerio de Salud Pública y Asistencia Social, Guatemala City, Guatemala
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  • Lissette Reyes
    Affiliations
    Área de Salud, Santa Rosa, Guatemala

    Global Disease Detection Branch, Division of Global Disease Detection and Emergency Response, Center for Global Health, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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  • Mary E. Wikswo
    Affiliations
    Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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  • Kyle F. Abramowicz
    Affiliations
    Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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  • Gregory A. Dasch
    Affiliations
    Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging Zoonotic Infectious Diseases, US Centers for Disease Control and Prevention, Atlanta, Georgia, USA
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  • Kim A. Lindblade
    Correspondence
    Corresponding author. Tel.: +1 404 718 4750; fax: +1 404 718 4815.
    Affiliations
    International Emerging Infections Program, CDC Regional Office for Central America and Panama, Guatemala City, Guatemala

    Malaria Branch, Center for Global Health, US Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS A06, Atlanta, GA 30333, USA
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  • Author Footnotes
    1 Jiann-Ping Hsu College of Public Health, Georgia Southern University, 501 Forest Dr., Statesboro, GA 30458, USA.
Open AccessPublished:December 20, 2012DOI:https://doi.org/10.1016/j.ijid.2012.11.011

      Summary

      Objectives

      We describe an outbreak of spotted fever group (SFG) rickettsiosis that occurred in 2007 in a farming community in southeastern Guatemala. We identified 17 cases of an acute febrile illness, among which 10, including two fatalities, were confirmed or probable cases of rickettsial disease (case–fatality proportion 12%).

      Methods

      PCR, a microimmunofluorescence assay (IFA), and Western blotting were performed on patient samples, and PCR was performed on ticks.

      Results

      Using an indirect IFA, seven of 16 (44%) ill persons tested had both IgM and IgG antibodies reacting with one or more Rickettsia spp antigens; the other nine (56%) had only IgM titers or were seronegative. Antibodies to SFG protein and lipopolysaccharide were detected by Western blotting with antigens of Rickettsia typhi, Rickettsia rickettsii, and Rickettsia akari. Only one sample, from an ill person who died, tested positive by PCR for a SFG Rickettsia. PCR analysis of Amblyomma cajennense ticks from domestic animals in the area detected the presence of SFG Rickettsia DNA in one of 12 ticks collected.

      Conclusions

      Further studies in Guatemala are warranted to establish the prevalence of rickettsioses and to fully characterize the identity of the etiologic agents and vectors.

      Keywords

      1. Introduction

      In the Americas, several rickettsiae of the spotted fever group (SFG) are endemic and pathogenic. The best known of these are Rickettsia rickettsii, the cause of Rocky Mountain spotted fever (RMSF), Rickettsia akari, the cause of rickettsialpox, and Rickettsia felis, the agent of flea-borne spotted fever.
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      Rocky Mountain spotted fever from an unexpected tick vector in Arizona.

      Secretaria de Vigilância em Saúde. Surto de Febre Maculosa no municipio de Petropolis – RJ. Rio de Janeiro, Brazil: Ministry of Health; 2005. Available at: http://portal.saude.gov.br/portal/arquivos/pdf/nota_maculosa_corrigida.pdf (accessed November 29, 2010).

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      An increase in human cases of spotted fever rickettsiosis in Yucatan, Mexico, involving children.
      Recently, another species of SFG, Rickettsia parkeri, has been found to cause an eschar-associated rickettsiosis in the USA and in South America.
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      Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever.
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      The application of more versatile and accurate molecular tools for the identification of rickettsiae from human specimens and ectoparasite samples has permitted the identification of new rickettsial agents and new tick vectors implicated in the transmission of various rickettsiae, thus expanding our knowledge of both the types of disease caused by rickettsiae and the geographic range of rickettsioses.
      Although R. rickettsii causing RMSF has been found in Mexico, Costa Rica, and Panama, where the disease is known as ‘fiebre manchada’,
      • Bustamante M.E.
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      • de Rodaniche E.C.
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      An apparent outbreak of rickettsial illness in Costa Rica, 1974.
      the presence and characteristics of rickettsiae in Guatemala have not been well studied.In 1898, an outbreak of epidemic louse-borne typhus was reported from Quetzaltenango, which continued to experience small outbreaks of this disease between 1943 and 1956.
      • Romero A.
      • Zeissig O.
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      • Rizzo L.
      Exanthematic typhus in Guatemala.
      Vaccination, sanitation, and louse-control measures apparently eliminated epidemic typhus by 1959.
      • Romero A.
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      • Rizzo L.
      Exanthematic typhus in Guatemala.
      • Cabrera J.I.
      • McAnally Jr., W.J.
      • Montoya J.A.
      Report on the activities in the control of typhus in the Republic of Guatemala, 1946–51.
      Subsequent surveillance identified epidemic typhus in several municipalities of Quetzaltenango between 1970 and 1972;
      • Romero A.
      • Zeissig O.
      • España D.
      • Rizzo L.
      Exanthematic typhus in Guatemala.
      however, some of these later cases may have represented recrudescent Brill–Zinsser disease. To date, neither SFG rickettsiae nor clinical cases resembling RMSF have been reported from Guatemala.
      We describe an outbreak of febrile illness in the peri-urban farming community of Moyuta in the Department of Jutiapa, Guatemala. The outbreak occurred from March to May 2007 and resulted in illness in 17 persons, with two deaths. Here, we present evidence that 10 of the 17 patients apparently suffered from a rickettsial disease.

      2. Materials and methods

      2.1 Epidemiological investigation

      Moyuta is a peri-urban community approximately 80 km from the capital, Guatemala City, in the department of Jutiapa on the border with El Salvador, situated at 1283 m above sea level (Figure 1). The maximum daily temperature averages 34 °C and minimum temperature 21 °C, with minimal seasonal variations. Total annual rainfall averages 1400 mm per year, with one rainy season between May and October. The small town of 17 499 inhabitants is surrounded by pastures and farmland and is served by one health center. On May 16, 2007, the Health Area of Jutiapa was informed of the second of two deaths in the previous month from a febrile illness of unknown origin among employees of a dairy farm. The Guatemala National Health Laboratory performed preliminary testing, and the presence of significantly high levels of IgG and IgM antibodies reacting to Rickettsia typhi and R. rickettsii antigens was detected using an indirect microimmunofluorescence assay (IFA) in the blood of four ill persons, including the first fatality (Castillo, unpublished data). To identify the etiologic agent and prevent further cases, the Guatemalan Ministry of Public Health and Social Welfare (Ministerio de Salud Pública y Asistencia Social, MSPAS), along with the Health Area of Jutiapa and the Centers for Disease Control and Prevention (CDC) Regional Office for Central America and Panama, initiated an investigation to identify additional cases. This effort involved identifying additional suspect cases, collecting appropriate biological samples from ill persons and domestic and wild animals, collecting ectoparasites, and conducting laboratory diagnostics.
      Figure thumbnail gr1
      Figure 1Guatemala and neighboring countries. The location of Moyuta is shown on the map.
      For the purposes of this outbreak investigation, a case was defined as fever and headache in a resident of the neighborhood surrounding the dairy farm (population 763) between March 1 and May 16, 2007. Household interviews were conducted in all 197 houses in the neighborhood using a questionnaire addressing type and duration of symptoms, demographic characteristics of the ill persons and their occupations, and their domestic, peri-domestic, or occupational exposure to pets, domestic animals, rodents, and ectoparasites.

      2.2 Collection of specimens from ill persons

      When possible, acute and convalescent ethylenediaminetetraacetic acid (EDTA) whole blood, serum and/or plasma were obtained from each resident meeting the case definition. For each serum sample, an aliquot was shipped to the Guatemala National Health Laboratory (Guatemala City, Guatemala) and the remainder was frozen at −70 °C and shipped to the CDC, Atlanta, Georgia, USA, for further testing.

      2.3 Collection of ectoparasites and animal samples

      Domestic animals (cows, horses, dogs, and cats) were examined for ticks and fleas; ectoparasites were collected and refrigerated in cryovials until they could be frozen at −70 °C and shipped to CDC in Atlanta. Flagging for ticks was conducted in the fields surrounding the dairy farm where the two deceased individuals had worked, as well as in the cow barns where they milked cows. One hundred live traps were placed around the farm to capture small mammals.

      2.4 Serological procedures

      Seroreactivity to rickettsial antigens was evaluated with an IFA assay conducted according to standard procedures, and further confirmed by Western blotting (WB). R. rickettsii strain Sheila Smith and R. typhi Wilmington grown in embryonated chicken eggs and R. akari MK cultivated in African green monkey kidney (VERO) cells were used as antigens, as previously described.
      • Paddock C.D.
      • Koss T.
      • Eremeeva M.E.
      • Dasch G.A.
      • Zaki S.R.
      • Sumner J.W.
      Isolation of Rickettsia akari from eschars of patients with rickettsialpox.
      For the IFA, fluorescein isothiocyanate (FITC)-labeled, goat anti-human IgG (gamma-chain specific) and anti-human IgM (mu-chain specific) antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were used at dilutions of 1:200 and 1:100, respectively. For the detection of IgM antibodies, IgG was absorbed with a Mini Rapi-Sep-M kit in accordance with the manufacturer's instructions (Panbio Diagnostics, Columbia, MD). Samples were tested at consecutive twofold serum dilutions starting at 1:32, and titers were represented as the reciprocal of the last dilution exhibiting reactivity with a specific antigen. IgG and IgM titers ≥64 were considered positive for this investigation.
      For WB, rickettsial cells purified from host cell debris were solubilized at room temperature and separated through 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, as described previously,
      • Paddock C.D.
      • Koss T.
      • Eremeeva M.E.
      • Dasch G.A.
      • Zaki S.R.
      • Sumner J.W.
      Isolation of Rickettsia akari from eschars of patients with rickettsialpox.
      and were transferred to 45 μm nitrocellulose membranes (Schleicher and Schuell Bioscience Inc., Keene, NH). Non-specific protein binding sites were blocked with 5% non-fat dry milk, followed by incubation with serum diluted at 1:100 and then with horseradish peroxidase (HRP)-labeled goat anti-human IgG (gamma-chain specific) and IgM Fc5 μ antibody (Chemicon-Millipore) at 1:2500 dilution. Bound enzyme was detected by reaction with substrate solution containing 0.015% 4-chloro-1-naphthol (Bio-Rad, Hercules, CA), 0.015% hydrogen peroxide, and 16.7% methanol in 0.02 M Tris-buffered solution (pH 7.5). The developed membranes were washed in water, dried between filter papers, and digitized. All serological tests were repeated twice and identical results were obtained on different dates by two different technicians.

      2.5 PCR analysis of human specimens

      DNA was extracted from 200 μl of EDTA-whole blood using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA). When blood specimens were not available, serum or plasma was centrifuged for 10 min at 14 000 × g and the resulting pellet, suspended in 200 μl of homologous supernatant, was used for DNA extraction. PCR assays for the rickettsial 17 kDa antigen gene were used for the detection of spotted fever and typhus group Rickettsia DNA using Amersham Ready-to-Go-Beads (Amersham Biosciences, Piscataway, NJ) or Qiagen Master Mix (Qiagen), as described elsewhere.
      • Paddock C.D.
      • Finley R.W.
      • Wright C.S.
      • Robinson H.N.
      • Schrodt B.J.
      • Lane C.C.
      • et al.
      Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever.
      • Massung R.F.
      • Davis L.E.
      • Slater K.
      • McKechnie D.B.
      • Puerzer M.
      Epidemic typhus meningitis in the southwestern United States.
      Semi-nested amplification of the 70–602 nt fragment of the rickettsial outer membrane protein (OmpA) gene was performed for species identification of the SFG rickettsiae.
      • Eremeeva M.
      • Bosserman E.
      • Demma L.
      • Zambrano M.
      • Blau D.
      • Dasch G.
      Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona.
      Genomic DNA of Rickettsia prowazekii, Rickettsia slovaca, or R. rickettsii was used as a positive control for the PCR assays. Amplification of ompB, sca4, and gltA was performed as described elsewhere; amplified fragments were sequenced and analyzed as described previously.
      • Eremeeva M.
      • Bosserman E.
      • Demma L.
      • Zambrano M.
      • Blau D.
      • Dasch G.
      Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona.
      Sequences generated in this study were submitted to the NCBI GenBank under the following accession numbers: 17 kDa protein gene: JF437645; OmpA gene: JF437646.

      2.6 Laboratory confirmed case definitions

      A clinical case of acute febrile illness was considered a probable case of rickettsiosis if there was a single positive titer from either acute or convalescent serum. A confirmed case of rickettsiosis was a positive PCR result or a fourfold increase in titer between acute and convalescent sera.

      2.7 Testing of tick specimens

      Ticks were identified to the genus using standard taxonomic keys.

      Barros-Battesti DM, Arzua M, Bechara GH. Carrapatos de Importancia Medico-Veterinaria de Regiao Neotropical. Um guia ilustrado para identificacao de especies. Sao Paulo: Vox/ICTTD-3/Butantan; 2006.

      Morphological identification of Amblyomma cajennense ticks was confirmed by direct sequencing of the mitochondrial 12S ribosomal DNA gene (GenBank accession number JF437644), as described previously.
      • Eremeeva M.E.
      • Zambrano M.L.
      • Anaya L.
      • Beati L.
      • Karpathy S.E.
      • Santos-Silva M.M.
      • et al.
      Rickettsia rickettsii in Rhipicephalus ticks, Mexicali, Mexico.
      Amblyomma tick DNA was extracted using the QiaMiniAmp Kit (Qiagen) following the manufacturer's instructions, eluted with 200 μl AE elution buffer and stored at 4 °C before testing. Rhipicephalus microplus DNA was extracted using the Wizard SV 96 Genomic DNA Purification System (Promega, Madison, WI) and a Biomek 2000 Robotic Workstation (Beckman Coulter, Fullerton, CA), and was eluted with 120 μl sterile nuclease-free water.
      • Bermudez S.
      • Eremeeva M.
      • Karpathy S.
      • Samudio F.
      • Zambrano M.
      • Zaldivar Y.
      • et al.
      Detection and identification of rickettsial agents in ticks from domestic mammals in eastern Panama.
      PCR testing for typhus and SFG rickettsiae was performed as described above.

      2.8 Cell culture isolation of rickettsiae

      Frozen tick samples were thawed and their triturates were inoculated into a 25-cm2 flask containing a confluent monolayer of Vero E6 cells (CRL 1587/Vero 76, American Type Culture Collection, Manassas, VA). Cells were maintained in RPMI-1640 cell culture medium (Gibco, Invitrogen Corp., Grand Island, NY) supplemented with 2% fetal bovine serum (HyClone Laboratories Inc., Logan, UT), 5% tryptose phosphate broth, and 1 mM l-glutamine at 32 °C and 34 °C for several passages.
      • Eremeeva M.
      • Bosserman E.
      • Demma L.
      • Zambrano M.
      • Blau D.
      • Dasch G.
      Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona.
      The presence of rickettsiae was monitored by PCR and microscopic observation of acridine orange-stained smears of the cultures.
      • Eremeeva M.
      • Bosserman E.
      • Demma L.
      • Zambrano M.
      • Blau D.
      • Dasch G.
      Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona.

      3. Results

      3.1 Epidemiological and clinical case findings

      Seventeen cases including two fatalities (case–fatality proportion 12%) were identified between April 10 and May 15, 2007, with illness onset dates from March 15 through May 15 (Table 1). In addition to headache and fever, ill persons also reported chills (76%), nausea (44%), abdominal pain (31%), vomiting (25%), and rash (31%). Symptoms persisted 1–20 days (median 4 days) following the onset of illness. A petechial rash with unspecified location was present during the illnesses of the two persons who died (residents 5 and 13) for 6 and 2 days, respectively. Similarly, petechiae were observed for 5 days in another adult resident (resident 4) who recovered; two ill children (residents 6 and 8) developed a maculopapular rash lasting for 9 and 20 days, respectively, that persisted for several days following resolution of fever. Eschar or eschar-like lesions were not reported for the two persons who died, nor were they noticed by the other patients in their retrospective self-reporting.
      Table 1Clinical and epidemiological characteristics of ill persons from Moyuta, Department of Jutiapa, Guatemala
      Patient IDSexAgeOccupationDate of onsetSymptoms (days of duration)
      Duration of symptoms is provided if records were available.
      Sought medical attention/ hospitalization (days)Treatment (days)Exposure factorsOutcome
      R, recovered; D, died.
      Category
      P (probable): single positive serology test or convalescent positive IgG and IgM serology with at least one antigen; C (fatal confirmed): PCR-positive; N (recovered suspect): IFA-negative or only IgM-positive serology.
      FeverHeadacheChillsMyalgiaNausea/ vomitingRashAbdominal painAnimal contactArthropod contact
      1F24Teacher3/15/2007Yes (4)Yes (5)YesYesNoneNoneYes (5)Yes/noAmoxicillin, acetaminophenDogs, cats, ratsNRRP
      2F68Housewife3/25/2007YesYesYesNoNoneNoneYesNRNRRatsNRRP
      3M34Farm worker4/10/2007Yes (3)Yes (3)Yes (2)NoNo/yes (4)NoneNoneYes/noAmoxicillin (2)Rats, dogs, chickens, cowsFlea bitesRP
      4F39Housewife4/10/2007Yes (5)Yes (3)Yes (6)YesNonePetechial (5)NoneYes/noNoneDogs, cats, chickens, sheepFlea bitesRP
      5M36Farm worker4/13/2007Yes (4)Yes (6)NoneNRNo/yes (1)
      Patient 5 also experienced diarrhea lasting for 46h.
      Petechial (6)NoneYes/noAmoxicillin (1 dose)Rats, dogs, chickenFlea bitesDP
      6F2Child4/19/2007Yes (7)Yes (7)Yes (7)NoYes (1)/noMaculopapular (9)NoneYes/noAcetaminophenRats, dogs, chickensNoneRP
      7M23Farm worker4/20/2007Yes (4)Yes (5)Yes (5)NoYes (4)/noNoneYes (4)No/noAspirin (8)Dogs, ratsNRRN
      8M8Student4/20/2007Yes (18)Yes (12)Yes (9)NoYes (4)/yes (4)Macular (20)Yes (6)Yes/yes (9)Penicillin, erythromycin, doxycycline (9)Dogs, chickens, ratsNRRP
      9M78Farm worker4/22/2007Yes (18)Yes (6)Yes (6)YesNoneNoneYes (6)No/noAcetaminophenRats, dogs, chickensNRRP
      10M7Student4/28/2007Yes (16)Yes (4)NoneYesNoneNoneNoneYes/noTrimethoprim (7)RatsNoneRN
      11M23Farm worker5/2/2007Yes (15)Yes (4)NoneNRYes (4)/noNoneNoneNo/noNoneRats, dogsNRRN
      12F18Housewife5/5/2007Yes (2)Yes (2)Yes (2)Yes (2)NoneNoneNoneYes/noAcetaminophenDogsNoneRN
      13M17Farm worker5/5/2007Yes (9)Yes (9)Yes (5)NoYes (6)/yes (6)Petechial (2)NoneYes/yes (1)Doxycycline (1 dose)Dogs, cowsFlea bitesDC
      14F47Housewife5/9/2007YesYesYesYesNoneNoneNoneNRNRDogs, pigsNRRN
      15F18Librarian5/12/2007Yes (5)Yes (2)Yes (3)NoYes (2)/noNoneNoneYes/noDiclofenac, ibuprofenRatsFlea bitesRN
      16F28Housewife5/12/2007Yes (2)Yes (3)Yes (1)NRNRNRNRYes/noDoxycycline (3)Rats, dogs, chickensNRRP
      17F11Student5/15/2007Yes (2)Yes (3)NoneYes (3)Yes (1)/noNoneNoneYes/noDoxycycline (2), acetaminophenRats, dogs, chickensFlea bitesRN
      F, female; M, male; NR, no records available.
      a Duration of symptoms is provided if records were available.
      b R, recovered; D, died.
      c P (probable): single positive serology test or convalescent positive IgG and IgM serology with at least one antigen; C (fatal confirmed): PCR-positive; N (recovered suspect): IFA-negative or only IgM-positive serology.
      d Patient 5 also experienced diarrhea lasting for 46 h.
      Of the 17 ill persons, eight (47%) were male, including the two fatalities. Their median age was 23 years (range 2–78 years). Six (35%) worked on the same dairy farm and 12 (71%) lived in the residential area of the farm. The two fatalities occurred in men who had been employed to milk the dairy cows; the second man had been hired to replace the first man after his death. Fourteen (82%) ill persons had known exposure to animals living on the farm, including cows, pigs, dogs, and chickens. Fifteen (88%) ill persons reported seeing rats during the day and/or evening hours or being bitten by fleas, or both. The presence of body lice or head lice was not reported by any ill person; however, respondents acknowledged being able to recognize ticks and seeing them routinely on animals. Twelve (80%) ill persons sought medical care at the local health center. Of these, eight received antibiotics, including amoxicillin (three patients), penicillin (one patient), erythromycin (one patient), trimethoprim (one patient), and doxycycline (four patients). One pediatric patient (resident 8) was hospitalized and treated with three different antibiotics. One of the ill persons who died (resident 5) was treated with amoxicillin, and the second (resident 13) received a single dose of doxycycline 1 h before he died. Five ill persons were given, or self-medicated with acetaminophen, diclofenac, ibuprofen, and aspirin; two ill persons recovered without medication.

      3.2 Testing of human specimens

      At the CDC, serum samples of seven (44%) ill persons tested positive by IFA for both IgM and IgG antibodies, reacting with antigens of R. typhi, R. rickettsii, and/or R. akari, with titers ranging from 64 to 4096 (Table 2). No IFA antibody was detected in two samples from days 5 and 7 post-onset for the second fatality (resident 13). The sample from the other fatality (resident 5) tested IFA-positive for IgG and IgM antibody to R. rickettsii antigen at the Guatemala National Laboratory (Castillo, unpublished data), but the sample was not available for confirmatory testing at CDC. Sera of seven tested ill persons (44%) had only low IgM titers to one or more antigens, and elevated IgM antibody reacting with R. akari antigen was only present in the serum of one of these persons (resident 16). IgG-reactive sera were collected at 22 to 46 days after the onset of the febrile illness and were classified as convalescent based on the time course of the illnesses. When all IFA-positive sera were tested by WB, the antibodies were primarily directed against high molecular weight surface protein antigens (≥110 kDa) of each of three rickettsiae (Figure 2, A and B). Some sera exhibited weak IgM antibody reactivity to R. akari and R. rickettsii lipopolysaccharide (LPS) antigens at approximately 37.4 kDa and strong IgG antibody reactivity against LPS as washboard at 84–32 kDa (Figure 2A). Negative and positive serum controls were processed for each serologic assay and they performed as expected.
      Table 2Laboratory assessment of serum and blood specimens for antibodies reacting against rickettsiae and for rickettsial DNA by PCR performed at the CDC
      Patient IDDays after onsetPCR
      P, plasma pellet; S, serum pellet; NT, not tested because of insufficient sample, no sample, or sample not suitable for PCR.
      IgM-IFA
      Reciprocal IgM and IgG titers against antigen of R. typhi (RT), R. rickettsii (RR), and R. akari (RAK).
      IgG-IFA
      Reciprocal IgM and IgG titers against antigen of R. typhi (RT), R. rickettsii (RR), and R. akari (RAK).
      RTRRRAKRTRRRAK
      156Neg (P)25651232<323264
      246Neg (P)512322561024<321024
      3NRNeg (S)12825664<32512<32
      431Neg (P)5121024128409620481024
      49NT1024512256322048256
      5
      Samples from patient 5 were not available for testing at the CDC.
      NRNTNTNTNTNTNTNT
      622NT128256<32512256128
      40NT646464<32128<32
      725Neg (P)326464<32<32<32
      825Neg (P)25612812832<32512
      38NT512641282563264
      924Neg (P)326464<32<32256
      1018NT<326432<32<32<32
      1116Neg (P)64<3264<32<32<32
      1218Neg (P)32<3232<32<32<32
      135Pos (P)3232<32<32<3232
      7Neg (S)32<3232<32<3232
      145Neg (P)643264<32<32<32
      152Neg (P)<326432<3232<32
      13NT646432<32<32<32
      162Neg (P)32<32256<32<32<32
      173Neg (P)32<32<32<323232
      16NT646432<32<3232
      CDC, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; NR, not recorded.
      a P, plasma pellet; S, serum pellet; NT, not tested because of insufficient sample, no sample, or sample not suitable for PCR.
      b Reciprocal IgM and IgG titers against antigen of R. typhi (RT), R. rickettsii (RR), and R. akari (RAK).
      c Samples from patient 5 were not available for testing at the CDC.
      Figure thumbnail gr2
      Figure 2Western blotting reactivity of representative serum specimens. Panels A and B show the two different patterns of Western blotting reactivity observed in IFA-positive persons: resident 4 (panel A) and resident 8 (panel B). Arrows indicate the positions of the protein molecular weight markers with the following sizes, from the top to the bottom: 116 kDa, 84.8 kDa, 53.9 kDa, 37.4 kDa, and 29.0 kDa (Bio-Rad). The following antigen abbreviations are used: A, R. akari; R, R. rickettsii; T, R. typhi.
      PCR was performed on 15 DNA samples from 14 ill persons whose blood samples were collected 2 to 56 days after the onset of illness (Table 2). Only the DNA from a precipitate of the serum of one of the fatalities (resident 13; day 5 after onset of the illness) tested positive for SFG rickettsiae using 17 kDa protein gene nested PCR (GenBank accession number JF437645). The nucleotide fragment of the fragment amplified had the greatest similarity (99%) with the highly conserved homologous gene fragments of the core SFG rickettsiae and differed from that of R. akari, R. typhi, and R. prowazekii. Other fragments typically used for molecular identification of Rickettsia, including gltA, ompA, ompB, and sca4, could not be amplified.

      3.3 Environmental assessment and testing of tick samples

      Five rats were trapped during the environmental investigation; they tested negative by PCR for Leptospira spp. at the Guatemalan National Laboratory (Castillo, unpublished data).
      A. cajennense and R. microplus ticks were collected from a horse and a cow, respectively, cared for by the two farm workers who died. No ticks were collected by flagging. Twelve A. cajennense and 85 R. microplus tested negative for DNA of typhus group rickettsiae. DNA from one A. cajennense tested PCR-positive for the ompA fragment of SFG rickettsiae. The nucleotide sequences of the ompA PCR amplicon had the greatest genetic similarity with ompA sequences of Rickettsia africae and Rickettsia sibirica cluster (GenBank accession number JF437646). Other fragments of Rickettsia, including 17 kDa protein gene, gltA, ompB, and sca4, could not be amplified from this tick DNA. Inoculation of the triturate of the PCR-positive tick into Vero cell cultures did not yield a rickettsial isolate following three consecutive blind passages at 32 °C and 34 °C. No fleas were collected during this investigation.

      4. Discussion

      To our knowledge, we report the first confirmed outbreak of spotted fever rickettsiosis in Guatemala. The diagnosis was made by identification of SFG Rickettsia DNA in a specimen from one of two persons who worked on the same dairy farm and who died of an acute febrile illness, and in a tick found on a farm animal. In total, 17 ill residents with similar clinical manifestations were identified from a small community in the Department of Jutiapa from March through May 2007. Evidence for significant rickettsial exposure (IgG IFA antibodies, PCR, WB) was obtained for 10 of them, including the two residents who died.
      Autopsies were not performed on the two deceased residents. Serum specimens were only obtained from one of the two deceased, and no specimens were available for testing at CDC from the first fatal case. The rickettsial etiology of this illness was confirmed by detection of rickettsial DNA in a specimen of serum collected on day 5 after onset of illness from the second fatality. This patient had not seroconverted to rickettsiae when he died on day 7 of illness. In contrast, IFA tests performed at the Guatemala National Health Laboratory on serum collected 5 days after onset of illness from the first fatality, detected high titers of antibodies reacting with both R. typhi and R. rickettsii. Negative testing for leptospirosis and ehrlichiosis in this patient and in other patients ruled out other etiologies responsible for this illness (not shown).
      Limitations of the samples available for analysis restricted confirmation of the specific rickettsial etiologic agent(s) of these illness(es). Laboratory results were obtained at CDC for 16 ill persons; however, standard paired acute and early convalescent serum specimens were available for only two ill persons (residents 15 and 17), who both exhibited only a weak IgM titer of 64 to R. typhi and R. rickettsii antigens. For other ill persons, only either acute or convalescent serum specimens were available. One DNA sample (from one of the fatalities) of the five DNA samples made from acute IFA-negative serum specimens tested positive by PCR for rickettsiae. Microimmunofluorescence (also called IFA) is presently the gold standard for the serological diagnosis of rickettsial diseases;
      • Chapman A.S.
      • Bakken J.S.
      • Folk S.M.
      • Paddock C.D.
      • Bloch K.C.
      • Krusell A.
      • et al.
      Tickborne Rickettsial Diseases Working Group
      CDC. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever, ehrlichioses, and anaplasmosis—United States: a practical guide for physicians and other health-care and public health professionals.
      however, its sensitivity depends on the timing of sample collection relative to illness onset. Serum samples obtained early in the infection are often negative or so close to the cutoff limit that results are difficult to interpret; this was the case for some ill persons in this investigation. The sensitivity of the IFA is estimated at 94–100% after 14 days of illness, and testing paired acute and convalescent samples substantially increases confidence in the results. In the case of less severe rickettsial infections, such as rickettsialpox and R. parkeri or R. africae infection, convalescent sera often do not demonstrate significant titers until 25–28 days after the onset of illness.
      • Paddock C.D.
      • Finley R.W.
      • Wright C.S.
      • Robinson H.N.
      • Schrodt B.J.
      • Lane C.C.
      • et al.
      Rickettsia parkeri rickettsiosis and its clinical distinction from Rocky Mountain spotted fever.
      • Fournier P.E.
      • Jensenius M.
      • Laferl H.
      • Vene S.
      • Raoult D.
      Kinetics of antibody responses in Rickettsia africae and Rickettsia conorii infections.
      The duration of antibody persistence after recovery from infection is also variable. In RMSF, IgG and IgM antibody titers increase concurrently by the second week of illness, and IgM antibodies disappear after 3–4 months, whereas IgG titers may persist from 7–8 months to several years.
      • Clements M.L.
      • Dumler J.S.
      • Fiset P.
      • Wisseman C.L.
      • Snyder M.J.
      • Levine M.M.
      Serodiagnosis of Rocky Mountain spotted fever: comparison of IgM and IgG enzyme-linked immunosorbent assays and indirect fluorescent antibody test.
      • Wilfert C.M.
      • Kleeman K.
      • Hicks J.L.
      • MacCormack J.N.
      • Anacker R.L.
      • Casper E.A.
      • Philip R.N.
      The incidence of Rocky Mountain spotted fever as described by prospective epidemiologic surveillance and the assessment of persistence of antibodies to R. rickettsii by indirect hemagglutination and microimmunofluorescence tests.
      Unfortunately, while we used R. typhi, R. rickettsii, and R. akari as surrogate antigens for the unknown etiologic agent(s) in this study and confirmed the presence of antibodies reacting with expected immunogenic rickettsial antigens in serum specimens by WB, it is not possible to define the rickettsial agent responsible for this seroconversion by serology, nor to exclude the possibility that titers were pre-existent in ill persons.
      • Hechemy K.E.
      • Raoult D.
      • Fox J.
      • Han Y.
      • Elliott L.B.
      • Rawlings J.
      Cross-reaction of immune sera from patients with rickettsial diseases.
      • Philip R.N.
      • Casper E.A.
      • Ormsbee R.A.
      • Peacock M.G.
      • Burgdorfer W.
      Microimmunofluorescence test for the serological study of rocky mountain spotted fever and typhus.
      In this outbreak, extensive cross-reactivity was observed with antigens of typhus and SFG rickettsiae by both IFA and WB. This could be due to a novel rickettsial species or to multiple exposures to a variety of rickettsial agents on different occasions during the residents’ lifetimes. Appropriate case–control studies and serological surveys are needed to characterize exposure to rickettsial agents found in different regions of Guatemala. We were not able to complete the molecular identification of the rickettsial agent causing this outbreak due to the lack of appropriate specimens such as whole blood, skin biopsy collected from the rash area, or autopsy specimens.
      Testing of arthropods collected from residents, pets, or the immediate environment of the suspected cases may allow the detection of rickettsial agents. Of the two tick species collected during this investigation, A. cajennense and R. microplus, only one Amblyomma tick tested PCR-positive for a SFG Rickettsia and it was genetically closely related to R. africae and R. sibirica rather than other more common species like Rickettsia amblyommii.
      • Bermudez S.
      • Eremeeva M.
      • Karpathy S.
      • Samudio F.
      • Zambrano M.
      • Zaldivar Y.
      • et al.
      Detection and identification of rickettsial agents in ticks from domestic mammals in eastern Panama.
      • Labruna M.
      Ecology of Rickettsia in South America.
      Until relatively recently, only A. cajennense, Haemaphysalis leporispalustris, and Rhipicephalus sanguineus were implicated as vectors for the transmission of R. rickettsii in Mexico and Central America.
      • Bustamante M.E.
      • Varela G.
      Estudios de fiebre manchada en Mexico: papel del Rhipicephalus sanguineus en la transmission de la fiebre manchade en la Republica Mexicana.
      • de Rodaniche E.C.
      Natural infection of the tick, Amblyomma cajennense, with Rickettsia rickettsii in Panama.
      • Fuentes L.
      • Caldern A.
      • Hun L.
      Isolation and identification of Rickettsia rickettsii from the rabbit tick (Haemaphysalis leporispalustris) in the Atlantic zone of Costa Rica.
      However, in Brazil and the USA, a more extensive list of tick species has been implicated in the natural cycles of many rickettsial agents pathogenic for humans.
      • Cragun W.C.
      • Bartlett B.
      • Ellis M.
      • Hoover A.
      • Tyring S.
      • Mendoza N.
      • et al.
      The expanding spectrum of eschar-associated rickettsioses in the United States.
      • Medina-Sanchez A.
      • Bouyer D.
      • Alcantara-Rodriguez V.
      • Mafra C.
      • Zavala-Castro J.
      • Whitworth T.
      • Popov V.
      • Fernandez-Salas I.
      • Walker D.
      Detection of a typhus group Rickettsia in Amblyomma ticks in the state of Nuevo Leon, Mexico.
      • Oliveira K.
      • Pinter A.
      • Medina-Sanchez A.
      • Boppana V.
      • Wikel S.
      • Saito T.
      • et al.
      Amblyomma imitator ticks as vectors of Rickettsia rickettsii, Mexico.
      • Shapiro M.
      • Fritz C.
      • Tait K.
      • Paddock C.
      • Nicholson W.
      • Abramowicz K.
      • et al.
      Rickettsia 364D: a newly recognized cause of eschar-associated illness in California.
      Beside A. cajennense, Amblyomma aureolatum and Amblyomma oblongogutatum are involved in the natural cycles of R. rickettsii, while R. parkeri has been detected in Amblyomma triste.
      • Labruna M.
      Ecology of Rickettsia in South America.
      Furthermore, recently reported SFG rickettsial agents causing R. parkeri-like illness in Brazilian patients from the Atlantic rain forest were found in Amblyomma ovale and R. sanguineus.
      • Sabatini G.
      • Pinter A.
      • Nieri-Bastos F.
      • Marcili A.
      • Labruna M.
      Survey of ticks (Acari: Ixodidae) and their Rickettsia in an Atlantic rain forest reserve in the State of Sao Paulo, Brazil.
      Small to medium size mammals are most frequently considered as reservoirs for many SFG rickettsiae. On the other hand, capybaras (Hydrochoerus hydrochaeris), the largest known rodent, are demonstrated to be a primary reservoir for R. rickettsii in Brazil.
      • Labruna M.
      Ecology of Rickettsia in South America.
      Also, a recent study in Mississippi, USA, suggested that cattle can serve as a transportation mode for the dissemination of R. parkeri-infected Amblyomma maculatum ticks.
      • Edwards K.T.
      • Goddard J.
      • Jones T.L.
      • Paddock C.D.
      • Varela-Stokes A.S.
      Cattle and the natural history of Rickettsia parkeri in Mississippi.
      Preliminary studies indicate that R. felis
      • Troyo A.
      • Álvarez D.
      • Taylor L.
      • Abdalla G.
      • Calderón-Arguedas Ó.
      • Zambrano M.L.
      • et al.
      Rickettsia felis in Ctenocephalides felis from Guatemala and Costa Rica.
      and Rickettsia hoogstraalii are also present in arthropods from Guatemala (Estevez and others, unpublished data). Based on experience in the Yucatan Peninsula,
      • Zavala-Castro J.
      • Zavala-Velazquez J.
      • Walker D.
      • Perez-Osorio J.
      • Peniche-Lara G.
      Severe human infection with Rickettsia felis associated with hepatitis in Yucatan, Mexico.
      • Zavala-Velazquez J.E.
      • Yu X.J.
      • Walker D.H.
      Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico.
      it is reasonable to expect that cases of cat flea rickettsiosis may also occur in Guatemala. Whether differences in the genetic or nutritional status of local human populations may also influence the clinical manifestations or morbidity of rickettsial diseases in Central America remains to be evaluated.
      In conclusion, we report the first confirmed outbreak of SFG rickettsiosis in Guatemala. Our data indicate a need for further rickettsial disease surveillance and education of the medical community about the signs and symptoms of rickettsial diseases and proper patient management, particularly when fever of unknown origin is present.

      Acknowledgements

      We thank Joseph Singleton for assistance with the IFA, Sandor E. Karpathy, Maria Zambrano, and Michele Sturgeon for laboratory assistance, Michael Levin for his contribution to the morphological identification of ticks, the CDC Biotechnology Core Facility Branch for synthesis of primers and cell culture, Edgar Manuel Santos and Rafael Sirai for assistance with the field investigation and rodent trapping, and David Moran for assistance with the preparation of figures. The National Hospital of Cuilapa was instrumental in the identification of the initial cases. Kyle F. Abramowicz and Mary E. Wikswo were supported by fellowships from the Oak Ridge Institute for Science and Education.
      The findings and conclusions described in this manuscripts are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention and the Department of Health and Human Services (MEE, MEW, KFA, GAD, KAL, ED).
      Conflict of interest: All authors declare no conflict of interest.

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