Corresponding Author: Flávia Sousa Gehrke, PhD, Laboratório de Parasitologia Experimental e Aplicada, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, Cidade Universitária, 05508-900 São Paulo, SP, Brazil. E-mail: firstname.lastname@example.org
Rickettsial spotted fever is common in southeastern Brazil. Differential diagnosis of pathogens can be performed with proper laboratory methods. A traveler arriving from Portugal developed a fatal febrile hemorrhagic syndrome diagnosed as spotted fever rickettsiosis. We isolated the agent, which was identified as Rickettsia conorii conorii by sequencing rickettsial genes.
Diseases caused by spotted-fever group Rickettsiae are important zoonosis and distributed worldwide. Rickettsiae are maintained in natural cycles involving several tick species, acting as their vectors and sometimes reservoirs, and vertebrate hosts present in a particular biotope. Global tourism has determined an increase in the number of travelers visiting remote areas with exposure to ticks.
Spotted-fever group Rickettsiosis generally begins as an acute undifferentiated febrile illness, often accompanied by headache, myalgia, and nausea, and a maculopapular or vesicular rash may be observed a few days after the onset of illness. Inoculation eschar is a typical clinical feature and a hallmark of tick-borne (TB) rickettsiosis, but it is absent in some diseases such as Rocky Mountain spotted fever caused by Rickettsia rickettsii in the Americas. The diagnosis of Rickettsiosis can be missed because of these nonspecific initial clinical presentations and the absence of specific laboratory confirmation. In Brazil, the TB disease Brazilian spotted fever (BSF) caused by R rickettsii and transmitted mainly by the horse tick Amblyomma cajennense, re-emerged at the end of the last century causing several fatal cases. In 2005, syndromic surveillances for febrile hemorrhagic diseases (dengue, measles, rubella, meningococcemia, staphylococcal syndromes, and rickettsiosis among others) carried out in the state of São Paulo to detect emerging diseases allowed us to diagnose a presumptive fatal case of Mediterranean spotted fever (MSF) caused by Rickettsia conorii, an agent known to be endemic in the old world only, and transmitted by the brown dog tick Rhipicephalus sanguineus.
In Portugal, MSF, also known as Boutonneuse Fever (BF), is a notifiable disease and usually recognized as a benign rickettsiosis. It can be usually treated in the outpatient setting, rarely having a severe or fatal course. The disease is characterized by a short onset of fever (>39°C), maculo-papular rash, inoculation eschar (“tache noire”) at tick bite sites, and myalgia. However, the number of MSF cases in Portugal is increasing, possibly as a result of climatic changes affecting vector seasonality, and also an increase of severe and fatal cases has been registered. In Portugal, R conorii conorii (formerly R conorii Malish) and R conorii israelensis (formerly Israeli tick typhus strains) are the agents of MSF.
In this work, we analyzed the nucleotide sequence of rickettsial genes detected in a Portuguese patient's blood clot in order to clarify the identity of the rickettsiosis agent. The protocol utilized in this research was approved by the Ethical Committee on Human Experimentation of the Instituto de Ciências Biomédicas da Universidade de São Paulo.
In July 2005, a 55-year-old Portuguese male was admitted to the Hospital das Clínicas of UNICAMP (State University of Campinas), a regional referral university hospital in Campinas municipality, state of São Paulo. The patient had arrived 3 days previously from Lisbon, Portugal. When initially examined in the emergency department he was alert, was febrile and had a petequial rash that rapidly evolved to a generalized hemorrhagic suffusion, and had shock, dying a few days later. The patient was a resident of Lisbon and days before traveling to Brazil he visited a peri-urban property with reports of the presence of dogs and risk of parasitism by ticks; there were no eschar or clinical signs of inoculation. He referred malaise and fever since departure, and presumptive diagnosis of spotted fever rickettsiosis was done at admittance and blood aliquot was collected.
The serum sample of the patient was analyzed using indirect immunofluorescence with antigens obtained from Vero cell-infected R rickettsii (Sheila Smith Strain). The antigens were prepared at the Adolfo Lutz Institute, São Paulo, Brazil. The IgM antibody titer ≥ 1:64 was considered positive. For culture, blood clot aliquot was centrifuged and the supernatant was inoculated in a confluent monolayer of Vero cells on circular slides adapted to the flat-bottomed tubes (shell vials). Infection of Vero cells was monitored by immunofluorescence reaction prepared with R rickettsii-positive human serum, which permitted us to observe the presence of fluorescent microorganisms in the form of intracellular bacteria, and SFG rickettsiae were isolated.
For molecular characterization of the agent, DNA was extracted from the patient's blood clot using QIAamp® DNA Blood (QIAGEN, Hilden, Germany), following the manufacturer's protocol. Rickettsial DNA was detected by polymerase chain reaction (PCR) using the previously described conditions and three sets of primers: CS-78 and CS-32, CS-239 and CS-1069, and Rr190.70p and Rr190.602n.[6, 7] The fragments were cloned into InsT/AcloneTM (Fermentas, Vilnius, Lithuania) and were sequenced in both forward and reverse directions using ABI Prism dGTP BigDye Terminator Ready Reaction Kit (Perkin Elmer, Foster City, CA, USA).
The partial sequences of rickettsial ompA and gltA genes were compared with corresponding sequences available in the GenBank (Figure 1). The sequences were aligned with the Clustal W software (1.60). To obtain a better alignment, both pairwise and multiple alignments parameters were changed from the default set. We used the DNA substitution matrix from the Clustal program, decreased the open gap penalty to 10, and also decreased the transition/transversion rate to 0.25. The alignments were used to construct similarity trees of nucleotide distances estimated by the Neighbor Joining algorithm and number of differences using the MEGA software (Molecular Evolutionary Genetics Analysis, version 3.01).
The PCR performed on DNA extracted from the patient blood sample yielded fragments with the expected lengths of gltA and ompA rickettsial genes. Partial sequence of gltA gene was 1,083 bp (GenBank access EU716648), and the nucleotide sequence of ompA gene fragment was 479 bp (GenBank access EU716649). The nucleotide sequences of ompA and gltA genes of our sample (R conorii ICB 1004) had more than 99% identity to the homologous sequences of three R conorii complex strains available in the GenBank. Similarity tree constructed based on ompA nucleotide sequences (Figure 1) clustered the strains of R conorii complex in a consistent clade within the R rickettsii group. Our sample was kept clustered together with R conorii conorii (formerly R conorii Malish strain), the agent of classic MSF, in a distinct clade from R conorii israelensis and R conorii caspia subspecies. The configuration of similarity tree constructed based on gltA was compatible with that of ompA.
The present diagnosis of R conorii conorii causing disease with a severe course in our patient confirms previous observations.[4, 5, 8, 9] Severe or fatal cases can be related to advanced age, underlying chronic diseases, or delay of appropriate treatment.[4, 8] Febrile hemorrhagic syndrome is a frequent manifestation of a wide variety of viral or bacterial infections, and a proper laboratory study to a precise identification of the agent is crucial. Rickettsial diseases have been frequently related in international travelers throughout the world in the last decades, most of them coming from sub-Saharan Africa.[1, 9, 10]
In Brazil, only one fatal case of spotted fever group rickettsiosis caused by R conorri conorii had been reported, in a South African traveler. This case is the first report of MSF in Brazil imported from Portugal, where R conorii is endemic. This study reinforces once more the importance of health surveillance in alerting local and tourism authorities to provide essential information to international travelers.
This reasearch was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Declaration of Interests
The authors state they have no conflicts of interest to declare.