Mediterranean Spotted Fever
and Identification of New Agents of
Rickettsioses in Portugal
Epidemiological Determinants, Host and Microbial Features in Portuguese Patients
RITA MARQUES DE SOUSA
Doutoramento em Ciências da Vida (Saúde Pública)
and Identification of New Agents of
Rickettsioses in Portugal
Epidemiological Determinants, Host and Microbial Features in Portuguese Patients
RITA MARQUES DE SOUSA
Supervisors
Professor Doutor Jorge Torgal
Departamento Universitário de Saúde Pública
Faculdade de Ciências Médicas da Universidade Nova de Lisboa
Professor David H. Walker
Department of Pathology
University of Texas Medical Branch, Galveston, Texas,
USA
Doutoramento em Ciências da Vida (Saúde Pública)
Ao Professor Doutor Jorge Torgal, pelo acompanhamento e orientação que sempre dedicou ao meu trabalho, não só durante a tese de Doutoramento mas em fases anteriores, como o da minha tese de Mestrado, pela amizade e pelo seu espírito crítico.
I would like to express my gratitude to Dr. David. H. Walker, the undisputed world leader in the field of Rickettsiology, who has guided me throughout my experimental work. He generously allowed me to perform a substantial amount of my work at his laboratory where I was received with open arms. His unabated dedication to mentoring my career is partially reflected in this dissertation.
À Doutora Sofia Núncio, e ao Professor Armindo Filipe pelo apoio institucional e confiança que me concederam enquanto responsáveis pelo Centro de Estudos de Vectores e Doenças Infecciosas.
À minha amiga Mestre Sónia Dória - Nóbrega pela incansável e sempre disponível colaboração. To Dra. Nahed Ismail for her friendship, for her critical supervision and availability to discuss troubleshoot and the little nuances of the laboratory work. She is magnificent immunologist who has shared openhandedly his knowledge and wisdom with me.
Aos médicos que colaboraram directamente comigo, e que incitaram na realização da presente investigação, em especial a Dra. Adelaide Belo, a Dra. Ana França, o Dr. Mário Amaro, o Dr. Tiago Tribolet de Abreu, o Dr. José Poças, a Dra. Paula Proença, o Dr. Pedro Costa, o Dr. Luís Duque, a Dra. Margarida Anes, a Dra. Teresa Ramos, a Dra. Graça Cristina, e a Dra. Graça Pombo.
A todos os meus colegas do CEVDI e em especial àqueles que trabalham comigo directamente, pela disponibilidade desinteressada, e devotado apoio.
To Donald Bouyer, Patrícia Valdes and all Walker lab residents thanks for the support during my stay at the laboratory and friendship.
Ao Pierre - Edouard Fournier com quem colaborei directamente enquanto realizei o meu estágio na Unidade de Rickettsias de Marselha.
Às Instituições
Este trabalho também não teria sido possível sem a colaboração de diversas instituições que a título profissional e/ou pessoal, apoiaram o presente estudo. Gostaria de agradecer
Ao Instituto Nacional de Saúde Dr. Ricardo Jorge pelas facilidades concedidas na realização desta tese de doutoramento.
Faculdade de Ciências Médicas da Universidade Nova de Lisboa por ter aceite a minha candidatura de doutoramento.
À “University of Texas Medical Branch, Department of Pathology,” por me ter aceite como investigadora e dado oportunidade de aí poder desenvolver os meus trabalhos.
À Fundação Luso Americana pelas bolsas concedidas no âmbito do meu trabalho que realizei na Universidade do Texas, nos Estados Unidos.
À Fundação Calouste Gulbenkian pela bolsa concedida na deslocação aos Estados Unidos. À Direcção Geral da Saúde, em particular à Dra. Judite Catarino por me ter disponibilizado dados de epidemiologia.
Ao ONSA/ Instituto Nacional de Saúde Dr. Ricardo Jorge, em particular ao Dr. Baltazar e Dr. Paulo Nogueira
Part I. Literature Review and Aims of the Dissertation Chapter I
Literature Review 3
01. Introduction 5
02. History 7
03. Etiologic Agent 11
04. Ecology and Epidemiology 19
05. Pathogenesis and Immune Response 25
06. Clinical Manifestations 33
07. Diagnosis 43
08. Treatment 49
09. Prevention 51
Chapter II
Aims of the Dissertation 53
Part II. Epidemiological Determinants Chapter III
Israeli tick typhus strain isolated from Rhipicephalus sanguineus ticks in Portugal 61
Chapter IV
Ticks and tick-borne rickettsial surveillance in Montesinho Natural Park, Portugal 67
Chapter V
Boutonneuse fever and climate change 75
Chapter VI
Portuguese patients 103
Chapter IX
Intralesional IFN-γ, TNF-α, IL-10, NOS2,indoleamine-2, 3-dioxygenase, and
RANTES are major immune effectors in Mediterranean spotted fever rickettsiosis 131
Part IV. Other Rickettsioses Chapter X
Rickettsia sibirica isolation from a patient and detection in ticks, Portugal 147
Chapter XI
Lymphangitis in a Portuguese patient infected with Rickettsia sibirica 151
Chapter XII
Molecular detection of Rickettsia felis, R. typhi and two new genotypes closely related
to Bartonella elizabethae in fleas from Portugal 163
Part V. General Discussion and Concluding Remarks Chapter XIII
General Discussion 173
Chapter XIV
Concluding Remarks 187
In the centennial of the first descriptions
Of boutonneuse fever, we continue enthusiastically
the study of this disease.
responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R. conorii Malish e R. conorii Israeli spotted fever strain. O principal vector e reservatório éo ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada com a de outros países da
bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/ 105 habitantes e 47,4/ 105 habitantes
respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de Inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica) e de estarem descritos casos graves em cerca de 5 - 6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento do número de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18%, respectivamente. Para além de factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição de terapêutica específica, foi colocada a hipótese de que a estirpe R. conorii Israeli spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomedamente que citoquinas, poderiamestar envolvidos na resposta a uma infecção por R. conorii.
virulenta do que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte em doentes com FEN. Associadas a factores de mau prognóstico da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.
Pakistan. In Portugal two strains cause disease: R. conorii Malish and R. conorii Israeli spotted fever.
Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season. The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectly the number MSF cases during the so called MSF off- season.
In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest
rates compared with other endemic countries. In Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with
47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this
pattern of the disease in Portugal has been changing. This fact became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admitted at Beja and Garcia de Orta hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portuguese patients in the same year (1997) causes different or atypical clinical manifestations that lead to a difficult diagnosis and/or this strain is more virulent compared to R. conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated. A prospective study was performed to characterize epidemiological, clinical, laboratory features and determined the risk factors for a fatal outcome.
One hundred forty patients (51% patients were infected with Rickettsia conorii Malish strain and 49% with Israeli spotted fever strain) with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portuguese hospitals during 1994-2006.
and immune mediators in skin biopsies collected from 23 untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO), an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2), a source of microbicidal nitric oxide. Positive correlations among TNF-α, IFN-γ, NOS2, IDO and mild- to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with severe MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression. Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.
In this dissertation is also described a new rickettsiosis present in Portugal caused by R. sibirica
mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy. The presence of this agent is corroborated by its detection also in Rhipicephalus pusillus
tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in
AFR Astrakhan fever rickettsia
ALT Alanine aminotransferase
AMP Adenosine monophosphate
APC Antigen - presenting cell
AST Aspartate aminotransferase
ATP Adenosine 5'-triphosphate
CA Cross-adsorption
CCL Chemokine
CD4 Cluster of differentiation 4
CD8 Cluster of differentiation 8
CEVDI Centro de Estudos de Vectores e Doenças Infecciosas
CO2 Carbon dioxide
CTL Cytotoxic T Lymphocytes
CXCL Chemokine
DCs Dendritic cells
DEBONEL Dermacentor-borne-necrosis-eschar-lymphadenopathy
DEET N, N- diethyl-m-toluamide
DGS Direcção Geral da Saúde
DIC Disseminated intravascular coagulation
DNA Deoxyribonucleic acid
DVT Deep venous thrombosis
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme – Linked Immunosorbent Assay
ESCMID European Society of Clinical Microbiology and Infectious Diseases
Fab Fragment, antigen binding
FEN Febre escaro-nodular
G6PD Glucose-6- phosphate dehydrogenase
gltA Citrate synthase
GMP Guanosine monophosphate
IFA Immunofluorescence Assay
IFN-γ Interferon
IL Interleukin
iNOS Indutible Nitric Oxide
INSA Instituto Nacional de Saúde
ISFR Israeli Spotted Fever Rickettsia
ITTR Indian Tick Typhus Rickettsia
LAR Lymphangitis associated rickettsiosis
LPS Lipopolysaccharide
MIF Microimunofluorescence
MSF Mediterranean Spotted Fever
NF- κB Nucleator factor - κB
NK Natural Killer
NO Nitric Oxide
NOS2 Nitric oxide synthase
ompA Outer Membrane Protein
ompB Outer Membrane Protein
PCR Polymerase Chain Reaction
pld phospholipase D
RANTES Regulated by activation, normal T-cell-expressed and secreted chemokine; CCL5
RMSF Rocky Mountain Spotted Fever
RNAm Messenger Ribonucleic acid
ROS Reactive oxygen species
SCA Surface Cell Antigen
SCID Severe Combined Immunodeficiency
SFG Spotted Fever Group
TBE Tick-Borne Encephalitis
TG Typhus Group
Th1 T- cell helper type 1
TIBOLA Tick Borne Lymphangitis
Part I.
01. Introduction
rickettsii (1906) of the spotted fever group and R. prowazekii of the typhus group, 15 named Rickettsia
species (mostly from the spotted fever group) were reported to cause disease in humans (Parola et al., 2005). However, it should be pointed out that half of these species have been isolated in the last 20 years. Furthermore, some species that were not considered to cause disease in man were recently described as pathogenic to humans. The most interesting example is R. parkeri, which was only considered pathogenic to humans 60 years after its first detection in Amblyomma americanum by R. R. Parker (Parker et al., 1939; Paddock et al., 2004). The opposite situation also occurs, i.e, the description of the disease many years before the isolation of the agent from human samples. For instance, African tick bite fever was described for the first time in southern Africa in 1911, although
R. africae was only isolated and taxonomically classified in 1992 (Pijper, 1934; Kelly et al., 1992). This recent boom in new or rediscoveries of Rickettsia species is undoubtedly related to the development of cell culture systems and the advent of molecular genetic technology. Likewise, the detection and identification of long known pathogens in patients from new and distinct geographic regions with diverse presentations has refuelled interest in these agents causing emerging infections and increased the awareness of clinicians. An example of this, occurred in 1997, when a new strain of R. conorii, only previously known in Israel, was isolated from a Portuguese patient, increasing the concern of Mediterranean spotted fever in Portugal (Bacellar et al., 1999).
02. History
Ricketts described the first vector-borne obligatory intracellular bacterium (Ricketts, 1909). S. Burt Wolbach established by histochemical detection the presence of the agent in endothelial cells in 1919, and Herald Cox (1938) propagated it in yolk sacs of embryonated chicken eggs. In 1909 Charles Nicole demonstrated the implication of body-lice as a vector of typhus (Gross, 1996). R. prowazekii was recognized as the etiologic agent by the two investigators H. Ricketts and S. Von Prowazek, who died of typhus. The name R. prowazekii was proposed for the first time by da Rocha-Lima in honor of these scientists (da Rocha – Lima, 1951).
The discovery of these rickettsiae led to subsequent descriptions of other rickettsioses.
Mediterranean spotted fever (MSF)
MSF was described by Conor and Bruch in Tunisia in 1910. These researchers working in Tunis, at the Pasteur Institute published a dispatch in the Society of Exotic Pathology, describing a new disease characterized by an abrupt, high fever, headache, chills and “elements dermo-epidermiques, s’enffacant en partie par la pression, de couleur rose ou rouge foncé, de la grosseur de une lentille… Elles sont difficiles
á classer dans le cadre nosologique… nit aches, ni des macules, ni des papules… de lésions boutonneuses”. This new nosological entity was referred to as fièvre boutonneuse de Tunisie.
The first description of the disease in Portugal was done clinically by Delfim Pinheiro in 1917, some years before those were reported in Italy by Carducci (1920) and Olmer in France (1925) (Azevedo, 1937). The Portuguese physician, a health delegate from Soure, carefully described the observation of several cases focusing on their seasonal occurrence and differentiating them from the exanthematic typhus cases occurring at that time “Devo frisar que os casos, em 1917, surgiram em Agosto e desapareceram em Setembro. Todos os demais teem sido observados no fim do Verão ou de Outono (….)
De modo que eu, não admito, até prova em contrário, que a doença que tenho observado e descrevi e o tifo
exantemático sejam uma e a mesma entidade mórbida (…) batizei-a de pintadôr por causa das duas características
clínicas mais salientes; mas os meus clientes chamam-lhe por corrupção de nome ou por acharem mais próprio,
However, it was only in 1930 with the publication of, La fièvre exanthématique (fièvre escarho-nodulaire) et son apparition au Portugal by Ricardo Jorge, that the presence of MSF was “accepted” by the scientific community. “Febre escaro-nodular” was the name designated by Ricardo Jorge based on clinical characteristics. “Nous l’appellerions volontiers fiévre escarho-nodulaire; nodulaire, pour marquer le type papuleux, lenticulaire, noueux de l’éxanthème; escarho, à cause de l’escarre initiale à l’affection” (Jorge, 1930a).
Subsequently to the first descriptions in Europe and during 20’s – 30’s decades, other clinical and epidemiological aspects of the disease were defined. Pieri and Brugeas of Marseille in 1925 reported the presence of a tache noire at the site of rickettsial inoculation by tick bite. In 1930 Durand and Conseil implicated the tick species Rhipicephalus sanguineus as the vector and reservoir of the disease in Northern Africa. Brumpt in his publication Longévité du virus de la fièvre boutonneuse (Rickettsia conorii) che tique, Rhipicephalus sanguineus, named the etiological agent Rickettsia conorii in honor of Conor (Brumpt, 1932).
03. Etiologic agent
According to the 9th edition of the Bergey’s Manual of Determinative Bacteriology, the genus
Rickettsia is classified in the α-proteobacteria subdivision, order Rickettsiales (that also includes the families Anaplasmataceae and Holosporaceae), and family Rickettsiaceae (Dumler et al., 2001). This family includes the genus Orientia along with Rickettsia (Tamura et al., 1995).
Rickettsia is a small (0.3-0.5 x 0.8 -1.0 μm) coccobacillary gram-negative, obligatory intracellular bacterium that resides free in the cytosol and occasionally in the nucleus of host cells (Anderson et al., 1965; Weiss, 1973). This lifestyle within a highly special niche, the eukaryotic cell, gave rise to unique adaptations, for example, the reduction of bacterial metabolism and the exploitation of host metabolites. Rickettsia has evolved several unique transport systems specific for large, charged metabolites, such as UDP-glucose, S- adenosylmethionine, ATP, NAD, UMP, GMP, and AMP (Winkler, 1976; Winkler & Daugherty, 1986; Winkler et al., 1999; Tucker et al., 2003; Schmitz-Esser
et al., 2004; Audia & Winkler, 2006). The ability to transport substrates that are present in the host cell cytosol and rarely available in the extracellular milieu has likely contributed to the evolution of the small-sized rickettsial genome (1.11-1.12 Mb), and many of de novo biosynthetic pathways characteristic of free-living bacteria are no longer present in rickettsiae (Anderson & Kurland, 1998).
R.typhi, the agent of murine typhus transmitted by the rat-flea; and the spotted fever group (SFG) that includes the majority of rickettsial species, transmitted by different arthropod vectors such as ticks, mites and fleas. This group includes 15 validated species of rickettsiae that have been described to cause disease in man (Table 1). The SFG rickettsiae were first grouped together because they were associated with ticks, had common antigenic features (Weiss & Moulder, 1984) and also because they all stimulated the formation of agglutinins to Proteus variabilis OX-19 strain in the Weil Felix test (Weil & Felix, 1916). Although the antigenic determinants of their immunological reactions were unknown, the distinctive immunogenic properties of rickettsial antigens were used in the first half of the century to distinguish among spotted fever rickettsiae. Cross-immunity and vaccine protection tests (Pijper, 1936) in guinea pigs, complement fixation (Plotz, 1943) or toxin neutralization tests (Bell & Stoenner, 1960) were successfully applied to the differentiation of R. rickettsii, R. sibirica, and R. conorii. Similarly, indirect microimmunofluorescence serologic typing with mouse sera was developed in 1978 and was used during long time as current reference method for the identification of new spotted fever group rickettsiae (Philip et al., 1978).
More recently molecular biology advances have helped to describe a high number of spotted fever group rickettsial species. However, although different phylogenetic studies using diverse rickettsial genes, such as 16S rDNA (Roux & Raoult, 1995), gltA (Roux et al., 1997), ompA (Fournier et al.,
R. sibirica Russian isolates North Asian tick typhus(Mill, 1936)) Asiatic part of Russia, China, Pakistan D. nuttali, D. marginatus, Haemaphysalis concinna, Ixodes persulcatus
R.sibirica mongolitimonae strain Lymphangitis-associated rickettsiosis (Raoult et al.,1996) Inner Mongolia, South Africa, France, Greece, Portugal, Spain Hyalomma asiaticum, H. truncatum, R. pusillus R. africae African tick bite fever (Pijper, 1934) Southern, Eastern and Western Africa, Reunion Island, West Indies A. hebraeum, A. variegatum
R. australis Queensland tick typhus (Andrew et al., 1946) Australia I. holocyclus, I. tasmani R. akari Rickettsialpox (Shankman, 1946; Huebner et al., 1946)) World wide Liponyssoides sanguineus
R. japonica Japanese spotted fever (Mahara et al., 1985) Japan, Korea I. ovatus, D. taiwanensis, Haemaphysalis longicornis, H. flava R. honei Flinders Island spotted fever (Stewart ,1991) Australia, Thailand Bothrocroton hydrosauri, I. granulatus, Haemaphysalis
novaeguineae R. heilongjiangensis Far Eastern spotted fever (1992) (Fan et al., 1999) Far East Russia H. concinna R. felis Flea-borne spotted fever (Schriefer et al.,1994) North , Central and South America, Europe Ctenocephalides felis R. slovaca TIBOLA / DEBONEL (Raoult et al.,1997; Lakos, 1997) Spain, France, Hungary D. marginatus, D. reticulatus R. aeschlimanni Unnamed (Raoult et al., 2002) Southern Europe, Morocco, South Africa H. marginatum
R. parkeri Unnamed (Paddock et al., 2004) USA, South America A. maculatum, A. americanum, A. triste
R. massiliae Unnamed (Vitale et al., 2005) Europe Rhipicephalus spp.
Figure 1. Relationships among the pathogenic SFG rickettsiae, inferred from sequence analysis of Sca1
gene. Phylogenetic analysis was carried out by maximum likelihood tree in TREE-PUZZLE program, using a quartet puzzling algorithm to generate the tree (adapted from Ngwamidiba et al., 2006).
R. conorii, the causative agent of Mediterranean spotted fever, is the most frequently isolated rickettsia and has the widest geographical distribution of the SFG rickettsial species. R. conorii
differences in virulence between these strains. The strains of this species can be distributed into four groups, to accommodate only genetically similar isolates. The first group includes the prototype strain R.conorii Seven or Malish and other strains such as Moroccan, Kenya, M-1, Simko, Manuel, Zim1, Zim C, 16-B, Spain96, URRCFrance, PoHuR1021, and PoTiR12. The second group includes the strains that are more similar to Israeli spotted fever rickettsia (ISFR): G-212; S- 484; A-828; R-293, PoTi28, PoHuR915, PoHu1450. The third group is formed by Astrakhan fever rickettsia (AFR): A - 167, Chad. Fourth group by the Indian tick typhus rickettsia (ITTR): VR – 597, that includes only isolates from ticks.
Figure 2. World distribution of R. conorii strains
two from ticks and one from a patient (Valero, 1949; Goldwasser et al., 1974). The disease that seemed initially restricted to that area is widespread in the Mediterranean basin, and in 1997 the first human isolate of ISFR was obtained in Portugal (Bacellar et al., 1999). More recently a retrospective study on characterization of Italian strains obtained from several MSF patients showed that ISFR had also been isolated from a Sicilian patient in 1991 (Giammanco et al., 2005 ). AFR was isolated from humans and ticks in the Astrakhan region and from a patient in Chad (Eremeeva et al., 1993; Fournier et al., 2003a). This strain was also detected by PCR in R. sanguineus collected in Kosovo (Fournier et al., 2003b).
The first descriptions of infections presumably caused by ITTR strain were made by Megaw in 1917. Subsequently other physicians have documented the disease based on clinical features and Weil-Felix test that may provide evidence of possible rickettsial infection but the test is nonspecific, and does not allow a definitive diagnosis (Mathai et al., 2001; Murali et al., 2001). More recently the use of specific serologic studies have confirmed the clinical cases (Jayaseelan et al., 1991, Parola et al., 2001; Sundhindra et al., 2004); however, until now, this strain has been only isolated from ticks (Robertson et al., 1970).
04. Ecology and Epidemiology
All spotted fever group rickettsiae are transmitted by ticks with the exceptions of R. akari and R. felis, which are transmitted by gamasid mites and fleas, respectively. Rhipicephalus sanguineus, commonly known as the brown dog tick, is the main vector and reservoir of R. conorii strains in the Mediterranean area, former USSR, northern Africa, and India (Table 1) (Rehacek & Tarasevich, 1988). However, R. conorii has also been isolated from other tick species. In Ethiopia (Philip et al.,
1966), Kenya (Heisch et al., 1962; Heisch et al., 1957), Zimbabwe (Kelly & Mason, 1990; Beati et al.,
1995) South Africa (Gear & Douthwaite, 1938), and northeastern of India (Rehacek, 1996). R. conorii seems to be transmitted by two other dog ticks, R. simus and Haemaphysalis leachi (Beati et al.,
1995). In 1994, Astrakhan spotted fever strain was also isolated from R. pumilio in the Astrakhan region (Eremeeva et al., 1994).
R. sanguineus found in Old World feeds on different hosts during its development cycle. Larvae and nymphs usually fed on small mammals, and adult
stage on
carnivores and large domestic and free living animals (Rehacek & Tarasevich, 1988; Gilot et al., 1990; Caeiro, 1992; Dias, 1994; Caeiro, 1999). In some situations, mainly in anthropozoonotic and domestic cycles, dogs are the main host for feeding all the stages of R. sanguineus (Gilot, 1984).infection for a later successful transovarian transmission (Burgdorfer & Brinton, 1975). Some studies have shown that transovarian transmission is more likely to occur when a female is infected in a previous stage, as a larva or nymph, rather than if a non-infected female feeds on an infected animal and transmits the rickettsiae to its eggs (Burgdorfer, 1967; Niebylski et al., 1999). Based on that, transstadial persistence seems to be very important to transovarian transmission, and the only way that rickettsia has to survive in nature, when climatic conditions are not favourable for the tick life cycle. Nevertheless, transstadial persistence may have different rates of success for different pathogenic rickettsiae. R. monacensis, very recently described as pathogenic to man, did not have so many deleterious effects on Ixodes scapularis tick tissues, and infection of ticks is not accompained by high mortality when compared with the other laboratory studies of transstadial transmission of R. conorii in R. sanguineus and R. rickettsii in Dermacentor andersoni (Niebylski et al., 1999; Santos et al.,
2002; Matsumoto et al., 2005a; Jado et al., 2007; Baldridge et al., 2007). Experimental studies do not reflect in total the natural cycle of rickettsiae in the tick hosts, and different factors related mostly to the process of the tick’s infection under laboratory conditions are not the same as occur in nature. However, the mortality caused by some pathogenic rickettsiae is evident and might explain why we find very few infected ticks in nature. In large surveys it was shown that the prevalence of R. rickettsii is less than one per 1,000 ticks, and a study published by Bacellar showed that from 3032 R. sanguineus collected in Portugal from different hosts (2282 Canis familiaris) only two isolates of R. conorii were obtained (Walker, 1989; Bacellar, 1999). A low prevalence (0.03 %) of R. conorii infection was also found in R. sanguineus in Castilla y León (Spain) (Fernández-Soto, 2003; Fernández–Soto et al., 2006). Nevertheless, and although a low prevalence of ticks are infected with pathogenic rickettsiae, many infected ticks survive until the adult stage, and these are the ones which are responsible for the success of transovarian transmission (Burgdorfer & Brighton, 1975). In fact, this low prevalence of tick infection with R. conorii described in Bacellar’s studies does not reflect the high incidence rate of MSF in Portugal (Bacellar, 1999).
hosts, and the co-feeding of ticks at the same site has been shown to increase feeding success and the transmission of some pathogens. This non-systemic transmission has been shown to be more successful in studies performed with viruses and spirochetes (Jones et.al., 1987; Labuda et al., 1997; Richter et al., 2002).
The role of the dog in the cycle of R. conorii is still not conclusive. The inapparent infection in dogs with R. conorii has been a controversial subject among researchers (Kulagin et al., 1960; Clerc & Lecomte, 1974). The concept that dogs can develop disease is strengthening with recent reports (Alexandre, 2005; Solano-Galego, 2006). Morever, it is interesting that the two strains of R. conorii
that exist in Portugal have been detected by PCR in blood of ill dogs in Portugal, (Alexandre et al.,
unpublished). Although, dogs cannot be considered the reservoirs of R. conorii because they do not develop a prolonged rickettsemia, we must not exclude their role totally because no studies have been reported (Kelly et al., 1992). The possibility remains that some ticks may be infected occasionally while feeding on infected dogs.
In the tropics and sub-tropics, R. sanguineus is found both indoors and out, but in urban situations dogs are virtually the only hosts of immature and adult tick stages. Dogs are capable of transporting seeking ticks to human habitations where these arthropods may bite man. R. conorii is transmitted to humans by inoculation of infected saliva into the wound while the tick feeds (Burgdorfer & Varma, 1967). However, it is important to bear in mind that humans are only incidental hosts and do not play a role in propagating the organism in nature (Rehacek & Tarasevich, 1988).
Incidence and seroprevalence of Mediterranean spotted fever
these cases were attributed to the introduction of imported vectors or transmission of infection in endemic areas (e.g., acquired during tourism activities) (Edlinger & Navarro, 1983; Walker, 2003). In some countries the incidence of this disease is unknown, and most of the knowledge is based on the human seroprevalence of antibodies to SFG rickettsiae. Endemic regions usually have a relatively high proportion of the population with antibodies against SFG rickettsiae, despite the absence of a prior clinical history of MSF. Serological surveys using immunofluorescence technique and R. conorii as antigen showed (in the Mediterranean region) a seroprevalence of 7.6 - 13.7% in southern Portugal (Bacellar et al., 1991), 18.3% in Negev (Israel) (Gross et al., 1983), 2-15% in Egypt (Botros et al., 1989), 5.6-7 % in Morocco (Meskini, 1995), 11.5% in Barcelona (Spain) (Espejo-Arenas et al., 1990), 26.3% in Sevilla (Spain) (Garcia-Curiel et al., 1984), 10.4% in Lazio (Italy) (Federico et al., 1989), 4.2% in Croatia (Radulovic et al., 1993), 20% in Slovenia (Novakovic et al.,
1991), 7.9% northern Greece (Daniel et al., 2002 ), 12% in Corsica (Raoult et al., 1985), 18% in southern France (Raoult et al., 1987), and 2.4 – 6.7 % in the Marseille region (Raoult et al., 1993). Inasmuch as the reporting of cases of MSF is not obligatory in all endemic countries, it is difficult to compare the incidence in infection of different regions, although in the last few decades there has been an increase in reported MSF cases in Portugal, Italy, Spain, France and Israel. It has been suggested that this increase might be related to climate change that influences tick activity (Walker & Fishbein, 1991; Gross et al., 1982; Mansueto et al., 1986; Segura-Porta et al., 1989; Scaffidi et al.,
In Portugal MSF has been an obligatory notifiable disease (ICD 10: A77.1) since 1950. Fluctuations in the incidence have occurred. Between January of 1989 and December of 2003, 12,818 MSF cases were reported to officials of the Portuguese Ministry of Health’s Department of Epidemiology (DGS), with an estimated annual incidence of 8.9/105 inhabitants. In this period the incidence of
MSF ranged from 14.5/105 inhabitants in 1991 to 4.3/105 inhabitants in 2003 (Sousa & Bacellar,
2003). Although the cases reported to the DGS have been decreasing since 1999, the number of patients hospitalized with MSF has increased and exceeded the number of the total reported cases in 2003. Based on laboratory confirmed cases, Sousa & Bacellar estimated that underreporting may reach 86% of the total cases (Sousa & Bacellar, 2004). The incidence rate in different regions of Portugal is very heterogeneous mostly due to oscillations in reporting and ecological factors related to the distribution and vector dynamics. In Portugal the two districts with the highest incidence are Bragança with 56.8/105 inhabitants and Beja with 47.4/105 inhabitants (Sousa et al., 2003a; Sousa &
Bacellar, 2004), which is a curious fact as they are geographically far apart districts and one in the north and other in the south.
In Italy during a 5- year period from 1998 to 2002, a national incidence rate of 1.6/ 105 inhabitants
was reported. However, if different regions are considered individually, the incidence is much higher in Sicily which accounts for 51.4% of all clinical cases, with an incidence rate of 9.3/105
inhabitants (Ciceroni et al., 2006). In France, a prospective study in the south of Corsica showed a higher incidence of 48/105 inhabitants compared with other regions (Raoult et al., 1985). Gross and
co-authors estimated that in Israel the annual incidence of MSF is 6.2/105 inhabitants with the
highest incidences of the disease found in western coastal area and the southern Negev Desert. In this region the incidence of rickettsial disease caused by R. typhi or spotted fever group rickettsiae can reach 20.8/105 inhabitants, with 65% attributed to spotted fever rickettsiae, and an annual
incidence of SFG rickettsiosis of at least 13.6/105 inhabitants is estimated (Gross et al., 1984,
Yagupsky et al., 1989).
nymphs and the painless experience of tick feeding make their detection more difficult when they are attached to the body (Raoult & Roux, 1997).
05. Pathogenesis and Immunity
Tick-borne Rickettsia is transmitted by inoculation of infected saliva into the wound. Tick attaches to the skin, and injects saliva that contains pharmacologically active substances that anesthetize preventing pain, modulate host defences and inhibit coagulation while the tick obtains its blood meal (Sonenhine, 1991; Brossard & Wikel, 2004). Most of the time, tick bite is painless and unnoticed, especially when small larvae or nymphs are involved. Tick bite is the main exposure route of rickettsial infection, but transmission of rickettsiae by crushing infected ticks and contamination by fingers or spills with infectious hemolymph through the mucosa (e.g., conjuntiva) have also been reported (Pacheco et al., 2000).
Rickettsiae can infect any type of nucleated cells, and the initial process after the tick bite and transmission of rickettsiae through the dermis might involve fibroblasts, macrophages, or dermal dendritic cells. In fact, there is still a large gap of understanding the initial target cells in the skin and the interaction between tick-transmitted rickettsiae and the host immune response at the site of inoculation (Walker & Ismail, unpublished). However, recent studies suggest that resident DCs might have an important role in innate and acquired immunity (Fang et al., 2007; Jordan et al., 2007).
to generalized vascular inflammation, an increase in microvascular permeability, and fluid leakage into the interstitial space, as a consequence of endothelial changes, such as the disruption of adherens junctions between infected endothelial cells, formation of stress fibers and the development of interendothelial gaps (Valbuena & Walker, 2005; Walker, 2007). Studies in vitro performed by Valbuena & Walker show that the formation of such gaps coincides with the change in endothelial cellular shape, from small polygonal to large spindle forms. The direct cause of the development of the interendothelial gaps during rickettsial infections has not been elucidated, but does not appear to be related to rickettsial stimulation of the polymerization of host actin. In vitro studies demonstrate that spotted fever group rickettsiae directly damage the host cells, and there is evidence that Rickettsia causes the endothelial cells to produce highly reactive oxygen intermediates (ROS), that are the principal cause of lipid peroxidation of the cell membranes and injury; (Eremeeva et al., 1998a; Eremeeva et al., 1998b; Eremeeva et al., 2001; Santucci et al., 1992). The oxidative stress-mediated injury of cultured endothelial cells is associated with depletion of host components such as glutathione and increased levels of catalase, which increases the concentration of hydrogen peroxide, and striking reduction in enzymes such as glucose-6-phosphate dehydrogenase, glutathione peroxidase, and catalase that are host defenses against ROS-induced damage (Silverman & Santucci, 1988; Hong et al., 1998).
Rickettsia - Host Cell Interaction
As stated above rickettsiae must enter host cells in order to grow and survive. To enter a cell, rickettsiae must attach to the surface of the mammalian cell, and some of the molecules and the mechanisms involved in this process have been described. Two major surface proteins of SFG rickettsiae, outer membrane proteins A (OmpA) and B (OmpB) have been recognized as the major ligands (adhesins) in the process of entry of Rickettsia, a critical step to establish a successful infection (Li & Walker, 1998; Martinez et al., 2004). Martinez and co-workers showed recently that
the presence of rickettsia and the ubiquitination of Ku70 through the ubiquitin ligase (c-Cbl) is required and plays an important role in the receptor-ligand interactions (Martinez et al., 2005). The process of R. conorii interactions with Ku70, the ubiquitination of Ku70 molecules recruited to the cell membrane, and other eventual proteins could generate a signalling cascade (i.e., signal transduction events) ultimately leading to the activation of the Arp2/3 complex. This complex regulated and activated by different pathways involving Cdc-42, phosphoinositide 3-kinase, c-Src family kinases, and tyrosine-phosphorylated proteins leads to localized actin cytoskeletal rearrangements that are necessary at the entry site, resulting in focal-induced phagocytsis of the rickettsia by the so-called zipper mechanism (Jeng et al., 2004; Walker, 2006).
Once located intracellularly, the phagosome vacuole in which Rickettsia enter is a potential death trap, if lysosomal fusion occurs and bactericidal mechanisms are activated. So, the only way that
Rickettsia has to escape is lysing the phagosomal membrane and entry into the cytoplasm. The mechanism of phagosomal escape remains unknown, althought it has been hypothesized to be mediated by to proteins with potential membranolytic activities: hemolysin C (tlyC) and a phospholipase D (pld) (Renesto et al., 2003; Whitworth et al., 2005).
Figure 3. R. conorii moving inside the cytoplasm through the propulsive force
Actin polymerization, is the main driving force for SFG (with exception of R. peacockii) rickettsiae locomotion for intracellular and intercellular movement (Goldeberg, 2001; Simser et al., 2005) (Figure 3). In contrast to SFG rickettsiae that are released from infected cells via long thin filopodia into which the bacteria are proplelled by actin filaments, typhus group (R. prowazelii and short tails in R. typhi) rickettsiae spread via bursting of the massively infected host cell. The SFG Rickettsia that utilizes actin-based motility activates actin assembly through the Arp2/3 complex. The latter, requires activation by RickA, a protein expressed on the rickettsial cell wall that contains a carboxy terminal WH2 (Wiskott-Aldrich syndrome protein [WASP] Homology 2) domain, very similar to particular domains of WASP-family proteins (Gouin et al., 2004; Jeng et al., 2004). Arp2/3 is recruited to the rickettsial surface where it acts as a nucleator of actin polymerization and induces the formation of a network of long unbranched filaments in Rickettsia spp. actin tails. The actin filaments that are reminiscent of those present in filopodia push the rickettsia to the surface of the host cell, where the host cell membrane is deformed outward and invaginates into the adjacent cell (Van Kirk et al., 2000; Monack & Theriot, 2001).
Most rickettsiae multiply in the cytoplasm, but sometimes Rickettsia has the capacity to enter and multiply within the cell nucleus. Intranuclear organisms also assemble actin tails, which can grow to great lengths and wind through the nucleus. Unlike actin tails associated with cytoplasmic rickettsial organisms, tails associated with intranuclear Rickettsia are not fixed in space but instead move and may generate force on the nuclear membrane (Heizen et al., 1999).
Non-specific innate immune response, cytokines and chemokines involved in host defense against Rickettsia
(RANTES), and CXCL10 (IP-10) are dependent on the presence of TNF-α and IFN-γ. The latter ones act synergistically to activate RANTES and IP-10 through nuclear factor-κB (NF-κB) and interferon regulatory factor-1 transcription factors (Ismail et al., 2002). Sousa and collaborators showed also that mRNA of inducible nitric oxide synthase, TNF-α, IFN-γ, and RANTES is expressed in skin lesions from patients with Mediterranean spotted fever (Sousa et al., 2007). Higher plasma levels of these cytokines and chemokines have been found in patients with MSF and other rickettsioses (Kern et al., 1996; Mansueto et al., 1994; Jensenius et al., 2003; Vitale et al., 2001) In addition to the NO-killing mechanism, two other mechanisms (dependent on the type of infected cell line) such as production of hydrogen peroxide and limitation of availability tryptophan via its degradation by indolamine -2, 3-diogenase seem to be implicated in rickettsicidal activity (Walker et al., 1999; Feng & Walker., 2000; Sousa et al., 2007). Other proinflammatory cytokines secreted by HUVECs infected with R.conorii, IL-1, IL-6, and IL-8, appear to play an essential role in the innate responses and macrophage activation early in the infection (Kaplansky et al., 1995). Studies performed by Kaplansky and collaborators suggest that R. conorii induces IL-1α production by HUVECs, which in turn induces the expression of adhesion molecules and the secretion of chemoattractants, such as IL-8 via an autocrine mechanism. Additionally, IL-6 also secreted by R. conorii - infected HUVECs may mediate the acute phase protein production associated with MSF. IL-6 might be implicated in the local differentiation and proliferation of T lymphocytes, through its effects on IL-2 and IL-2 receptor in T cells. The role of chemokine induction, early in infection,
Acquired immune response
The immunity to primary rickettsial infections is characteristically a Th1 - type cellular immunity, and IFN-γ is an important component of this type immune response that effectively controls intracellular pathogens including rickettsiae (Walker, 2000; Biron and Gazzinelli, 1995). The cytokine IFN-γ in conjunction with cytotoxic T-lymphocyte activity and other cytokines has been shown to be very important for the elimination of different microorganisms (Walker et al., 2001). IFN-γ and CD8+ T cells are of crucial importance in rickettsial clearance, and it was shown that IFN-γ gene knockout mice were more than 100-fold more susceptible to R.australis infection than wild-type C57BL/6 mice (Walker et al., 2001).
Rickettsial immunity is mediated in part by CD4+ T cells, but clearance of spotted fever group rickettsiae from endothelial cells requires immune CD8+ T lymphocytes. In fact CD8+ T lymphocytes are essential in the effective immune response against rickettsiae. Feng and collaborators showed that an ordinarily sublethal dose of R. conorii is lethal or causes persistent infection in mice that are depleted of CD8+ T lymphocytes. In contrast, mice depleted of CD4+ T cells clear the infection in a similar time frame as the control group (Feng et al., 1997, Valbuena et al., 2002). CD4+ T-cell-mediated protective immunity requires T-cell production of IFN-γ, whereas CD8+ T cells mediate protection via both IFN-γ production and cytotoxic killing (Feng et al., 1997; Walker et al., 2001). The importance of cytolytic mechanisms by cytotoxic T-lymphocyte is shown by R. australis infection of C57BL/6 perforin gene knockout mice, which are 1000-fold more susceptible to lethal infection with rickettsiae, than wild-type mice. This suggests that perforin activity accounts for some of the CTL-mediated anti-rickettsial effect (Walker et al., 2001; Valbuena
and the generation of Rickettsia - specific CD8+ T cells that lyse infected target cells via pathways involving perforin and/or granzymes (Ismail et al., 2002).
06. Clinical Manifestations
MSF is characterized by generalized endothelial infection of the microvasculature, and the main clinical features are due to the injury of blood vessels caused by rickettsial infection. The histopathological phenomenon of vasculitis can involve all the organs, not only the skin, and it has particularly serious manifestations when the lungs and brain are affected (Walker & Mattern, 1980, Walker et al., 1997). The incubation period ranges from 3 to 7 days after tick bite, but it can be longer, and this fact can be related to the size of the bacterial inoculum. During the first 2 to 3 days of disease, the observed symptoms are non-specific and difficult to distinguish from more common illnesses. Patients complain of severe headache, fever and myalgia, but other symptoms early in the course can include malaise, anorexia, chills, nausea, vomiting, conjunctivitis and photophobia. After this period, the disease is characterized by fever (91-100%) greater than 39ºC. The fever usually will decrease, and apyrexia will occur within 3-4 days after specific therapy. Severe myalgias mainly in the lower limbs, arthralgias, sweating, chills, gastrointestinal involvement with nausea, vomiting, abdominal pain, and diarrhea, hepatomegaly, splenomegaly, and conjunctivitis have been reported. The variable incidences of manifestations in different series of patients with MSF are described in Table 2. Between the 3rd and 5th febrile day, 72 – 100 % of patients develop a rash, which initially
remission of clinical symptoms. In severe cases, vascular damage can result in purpuric or petechial (5-10%) rash, which is generally indicative of a bad prognosis (Amaro et al., 2003; Raoult et al., 1986; Mouffok et al., 2006). Absence of rash has also been reported (Brouqui et al., 1992; Amaro et al.,
2003, Morais et al., 1996, Parra–Martinez et al., 2002). Patients may suffer other hemorrhagic manifestations such as epistaxis, hemoptysis, and upper gastrointestinal hemorrhage (Mansueto et al., 1983). Most patients with MSF also develop focal epidermal and dermal necrosis, an eschar or “tache-noire” as a consequence of endothelial injury caused by R. conorii, at the site of the tick bite (Walker et al., 1988). This sign appears as a black crust approximately 1 cm in diameter but can be diverse in shape; normally surrounded by an erythematous areola (Figure 4).
Figure 4. Eschar surrounded by an erythematous areola
(kindly provide by Dr. Mário Amaro, Hospital Garcia de Orta, Almada)
of Clinical Microbiology and Infectious Diseases, where a complete absence of eschar was noted in patients infected with ISF strain (Brouqui et al., 2004; Sousa et al., 2004; Giamanco et al., 2005). Infection caused by Astrakhan spotted fever rickettsia showed similar clinical manifestations as the other strains of R. conorii; however, eschar has been reported in only 23% of patients (Tarasevich et al., 1991). Multiple eschars have occasionally been described in patients infected with R. conorii
Malish strain (Font-Creus et al., 1985; Mouffok et al., 2006). The detection of the eschar is often difficult because it is hidden in some parts of the body such as the axilla, palpebra, groin, and submammary fold. In adults the eschar is more frequently found on the lower limbs, groin and axilla and abdomen compared with children, where the scalp is the most common site (Feigin et al.,
2007).
Other reported complications mostly associated with severe cases include central nervous system impairment (Botelho - Nevers et al., 2005; Balsera et al., 2007) including meningoencephalitis (Agba et al., 1994; Dolado et al., 1994) and encephalitis (Wolach et al., 1989; Parra –Martinez et al., 2002), acute renal failure (Beorchia et al., 1986; Donati et al., 1990), hepatic injury, hemolysis associated with G6PD deficiency (Raoult et al., 1986; Regev-Yochay et al., 2000), pneumonia (Gil-Llano et al.,
1985; Marcos-Sanchez et al., 1989; Bellissima et al., 2001), pulmonary thromboembolism (Brahim et al., 2006), myocarditis and pericarditis (Catón et al., 1998; Drancourt et al., 1991), oculoglandular syndrome (Espejo et al., 1988), and uveitis (Castanet et al., 1988; Donati et al., 1990; Agba et al.,
1994; Balsera et al., 2007). Most severe MSF cases result in septic shock with multiorgan failure and a rapid course with deaths within 24 hours of admission (Amaro et al., 2003; Yaguspky et al., 1993, Raoult et al., 1986, Herrero-Herrero, 1994, Beltrán et al., 1985). Coagulation disturbances in MSF seem to be related to severity of infection, and injury to vascular endothelium stimulates activation of the coagulation system, platelets, and fibrinolysis with frequent thrombocytopenia (Vicente et al.,
in peripheral gangrene of the fingers and toes in some cases of MSF, occasionally requiring amputation (Walker & Gear, 1985). MSF can be a severe illness, particularly in patients with underlying conditions such as diabetes mellitus, heart failure, chronic alcoholism, increasing age, and G6PD deficiency (Walker, 1990; Regev-Yochay et al., 2000; Sousa et al., 2003). The disease is milder in children, and few cases of severe or fatal cases have been described in childhood. Different pediatric patients series of MSF are described in table 3 (Gross & Yagupsky, 1987; Pares
et al., 1988; Wolach et al., 1989; Cascio et al., 1998; Colomba et al., 2006).
* values are in percentage, ** include 9 children, 4 adults
Sample (n) 227 164 199 13 246 80 110 144 15
Fever 100 100 100 100 100 98 96 100 100
Eschar 73 62 72 0 74 88 89 84 13
Rash 99 100 97 100 99 100 97 96 100
Myalgia 73 89 36 23 79 56 80 79 93
Arthralgia 73 68 69 43 79 79
Headache 69 78 56 84 85 85 76 87
Gastrointestinal
signs 37 36 41 71 24
Conjunctivitis 32 50 9 85 9 25 20
Hepatomegaly 44 31 13 23 29 26 13 13
Splenomegaly 19 16 6 46 9 4 7 13
Thrombocytopenia 35 8 6 8 17 33
Leucopenia 20 77 8 4 23 13
Leucocytosis 28 10 31 30 12 74
Hyponatremia 25 31 4 31 39
Elevation of
AST/ ALT 55/53 39 85 55/50 24/32 70/76 60/49 60
Fever 100 100 97 98 100 100 100 91 93
Eschar 53 70 88 39 52 77 56 20 59
Rash 87 72 100 100 90 100 97 93 97
Myalgia 60 73 55 43 65 24 27 59
Arthralgia 91 28 24 11
Headache 80 84 74 69 57 63 78 24 48
Gastrointestinal signs 31 17 20 31
Conjunctivitis 53 34
Hepatomegaly 4 16
Splenomegaly 1
Thrombocytopenia 54 32 49 80
Leucopenia 47 8 9 14
Leucocytosis 25 21 39 26
Hyponatremia 23 24 57
Elevation of AST/ ALT 80 61/52 44 84/84
location Beersheva, Israel Barcelona, Spain Kfar Saba, Israel Sicily, Italy Sicily, Italy
Sample (n) 54 130 70 645 415
Age (median) 5 years old 7 years old 7 years old 6 years old 6 years old
Fever 100 100 100 97 93
Eschar 0 87 0 72 63
Rash 100 98 98.5 96 95
Myalgia 33 54
Arthralgia 13
70
25 38
Headache 22 63 24 15
Gastrointestinal signs 28
Abdominal Pain 6 33 14 7
Nausea
Vomiting 31 28 40 4
Diarrhea 9
Hepatomegaly 30 16 10
Splenomegaly 35 8 34 66
07. Diagnosis
eruption may be highly informative. The rash of MSF, for example, most often begins on the lower extremities and then spreads centripetally, whereas most drug eruptions and viral infections begin on the face or trunk and spread centrifugally. The latter kind of rash distribution is also seen in infections caused by typhus group rickettsiae. The hallmark for MSF and some of the other spotted fever rickettsiae that may help in differential diagnosis is undoubtly the presence of the rash on the palms and soles. However, this does not occur in all rickettsial infections, and for example it has been reported that in R. rickettsii infection this distribution typically occurs late and in only half of the cases (Weber et al., 2005).
Laboratory diagnosis
The diagnosis of rickettsial disease is most often confirmed by serological tests because culture, PCR and other methods require specialized laboratories, and also usually serum is the most common available sample. Indirect immunofluorescent assay remains the gold standard serologic test and is the mostly widely used technique for diagnosis of rickettsial diseases (Figure 5) (Brouqui
et al., 2004).
Figure 5. Positive reaction by immunofluorescence assay
The diagnosis is confirmed by demonstration of rise in titer of antibodies to specific rickettsial antigens by comparing the titers of acute and convalescent sera. In approximately 97% of patients, the acute serum usually does not contain antibodies, and serologic evidence of infection occurs no earlier than seven days after the onset of illness (Sousa et al., 2007). According to guidelines of ESCMID study group and based on what the Unité des Rickettsies de Marseille determined, they considered the cut-off titers of IgG and/or IgM to be ≥128 in suspected cases of R. conorii
infection, whereas IgG titer ≥64 and/or IgM ≥ 32 are considered indicative of infection by other
1992; Hove & Walker, 1995; Paddock et al., 1999; Galvão et al., 2003; Sousa et al., 2005; Zavala-Castro et al., 2006).
Culture of the agent is the ultimate criterion to confirm the diagnosis and to identify the species of rickettsia from patient’s blood. This technique can reach a good level of success if the sample is collected and preserved under good conditions until isolation procedures. Isolation of rickettsiae is performed by shell –vial assay from heparinized blood (leukocytic cells in the buffy coat), or skin biopsy samples, before specific antibiotic therapy. The success rate of isolation at the Portuguese National Institute of Health is around 9%, 85 isolates from 914 blood samples received at CEVDI (1994-2005), with clinical suspicion of rickettsial infection, mostly MSF (Sousa, unpublished data). However, some of these samples are also request for isolation testing of Ehrlichia chaffeensis,
Anaplasma phagocytophilum and Coxiella burnetii, and the laboratory already had the experience of a positive isolation of C. burnetii from patients with clinical diagnosis of rickettsial infection (Santos et al., unpublished). The reported rate of Rickettsia isolation at the Unité of Rickettsie in Marseille (1991-2003) is around 3% corresponding to 15 isolations from a total of 510 inoculated blood samples with clinical diagnosis compatible with rickettsial infection (Gouriet et al., 2006). However, a higher percentage of isolations, about 11% (37/337) were obtained from skin biopsies. The isolation from skin has shown to be a good alternative to blood isolation, for different species of
Rickettsia (Gouriet et al., 2006; Paddock et al., 2006). Isolation from blood has been very important to confirm the diagnosis of MSF in fatal Portuguese cases. The rapidly fatal cases have such a high level of rickettsemia that nearly 100% of fatal cases yield isolates of the rickettsiae, whereas virtually no one at the time of admission has developed antibodies, and a second convalescent sample is not available in fatal cases (Sousa et al., 2007 unpublished).
The centrifugation shell-vial technique first developed for cytomegalovirus was then adapted for the isolation of rickettsiae (Marrero & Raoult, 1989). The success of this technique has been also applied to the isolation of other infectious agents (Jayakeerthi et al., 2006; Fournier et al., 1998; Gouriet et al., 2006). Different kinds of cells might be used for blood and skin rickettsial isolation such as epithelial kidney cells of green monkey (Vero), mouse fibroblast (L929), human embryonic lung fibroblasts (HEL) and human fibroblasts cells (MRC5) (Cardenosa et al., 2000; Gouriet et al.,
previouslylisted cells and cells from mosquitos, ticks, and toads (Ereemeva et al., 2006; Horta et al., 2006; Labruna et al., 2007).
08. Treatment
09. Prevention
Aims of Dissertation
In 1997, a reported case fatality rate of 32% in patients with Mediterranean spotted fever admitted at Distrital Hospital of Beja, caught my attention as a researcher working at the Rickettsial Unit of National Institute of Health. The high fatality rate of MSF seemed to be changing from the usual pattern of disease described by the Portuguese physician, Ricardo Jorge (1930), ‘Maladie extrèmement bégnin depourvue presque de tout gravité, elle ne serait qu’un sujet curieux d’etude...’ (Jorge., 1930) . Moreover, in the same year Bacellar and collaborators identified for the first time in Almada Hospital the causative agent R. conorii Israeli spotted fever strain in MSF fatal cases only known previously to be present in Israel (Bacellar et al., 1999). Until 1997, only R. conorii
Malish strain was isolated from Portuguese patients.
impossibility of identifying the strain involved in the disease in patients admitted to Beja’s Hospital. A hypothesis was considered, and it might be that the new ISF strain isolated from Portuguese patients causes different or atypical clinical manifestations that lead to a difficult diagnosis and/or may be this strain was more virulent compared to those caused by R. conorii Malish strain. Additionally, the tick species responsible for the transmission of ISF strain, and its geographical distribution in our country was not identified. Another interesting aspect of this study was the irregular and heterogeneous notification of the disease all over the country. The higher incidence rates of reports were detected in two districts, Bragança in the North of Portugal and Beja in the South. However, although Bragança was the district with higher incidence of MSF, only 3 fatal cases occurred in a period of ten years, compared with the case fatality rate of 4.5% occurring in Beja district in the same period. The possibility of the ISF strain not existing in the North of the country was suggested. On the other hand the climate variables did not help in understanding or tick density or the distribution in both districts, because even though they are very far from each other, they share similar climatic conditions. In fact, the number of admissions of severe MSF cases kept increasing after 1997, not only in Beja hospital but also in other hospitals of the country. The questions about the presence and virulence of ISF strain, the high incidence (8.4 /105 inhabitants)
of the disease in Portugal, and one of the highest incidence compared with other countries from the Mediterranean area kept inciting my interest and led me to try to extend my knowledge of this disease in Portugal. This PhD dissertation is a follow-up of the previous study performed about MSF in Portugal and an attempt to try to answer some remaining questions.
- Epidemiological Determinants
i) Identification and distribution of the possible vector tick species of R. conorii Israeli tick typhus strain in our country.
ii) Climate change and indirect implications in MSF cases. iii) Epidemiology of MSF cases in Portugal.
- Studies on Patients
i) Isolation and characterization of strains from patients with MSF.
ii) Correlation of epidemiological, clinical and laboratory features of patients infected with different strains of R. conorii.
iii) Evaluation of risk factors for fatal outcome in patients infected with R. conorii Malish versus Israeli spotted fever strain.
iv) Understanding the mechanisms of immune response against rickettsial infection in patients infected with Rickettsia conorii strains.
- Other Rickettsioses
i) Rickettsial isolation and clinical features of a new tick-borne rickettsiosis in Portugal. ii) Identification and characterization of new flea-borne rickettsial agents in Portugal.
