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Dipartimento di Igiene e Microbiologia
Dipartimento di Medicina Clinica e delle Patologie Emergenti, Universita di Palermo, Palermo, Italy
ABSTRACT
A retrospective analysis by molecular-sequence-based techniques was performed to correctly identify the etiological agent of 24 Mediterranean spotted fever cases occurring in Western Sicily, Italy, from 1987 to 2001. Restriction analysis of a 632-bp PCR-amplified portion of the ompA gene allowed presumptive identification of five clinical isolates as belonging to Rickettsia conorii subsp. israelensis, the etiological agent of Israeli spotted fever (ISF). The remaining 19 rickettsial isolates were Rickettsia conorii subsp. conorii, the only pathogenic rickettsia of the spotted fever group reported in Italy until the present. Sequence analysis of the ompA gene confirmed the identification of all the R. conorii subsp. israelensis isolates and demonstrated that rickettsiosis caused by R. conorii subsp. israelensis can be traced back to 1991 in Sicily. The recorded clinical data of the five ISF patients support the idea that these strains could correlate to more-severe forms of human disease. Three of five patients experienced severe disease, and one of them died.
INTRODUCTION
Mediterranean spotted fever (MSF), also known as boutonneuse fever, is caused by Rickettsia conorii, an obligately intracellular, slow-growing gram-negative bacterium. The disease usually has a benign course and is characterized by the onset of high fever, myalgia, arthralgia, and a typical rash. However, about 6% of the cases are severe, and fatal cases do occur even in young, healthy adults, with a reported death rate of about 2.5% (15). MSF is endemic in Italy, where it is a reportable disease. The Italian Ministry of Health received reports of 890 cases of human rickettsioses presumed to be MSF in 2002. MSF is more common in some central and southern regions of Italy (20, 22), reaching an average of 10 cases for every 100,000 inhabitants in Sicily in 2002, compared with a national average of 1.6. Rickettsia conorii is thought to be the only pathogenic rickettsia of the spotted fever group (SFG) in Italy (22), as well as in the Western Mediterranean area, even if the possible circulation of strains different from classical R. conorii has been proposed, mainly for clinical reasons (different degrees of severity) (24).
In recent years, the systematic identification of rickettsial species causing human infections has continued to increase the number of recognized human pathogens. Since 1984, several new agents of SFG rickettsioses have been recognized, including R. japonica, R. honei, R. africae, R. felis, R. slovaca, and R. aeschlimannii (9, 12-14). Unusual rickettsial strains related to R. conorii have been described as belonging to an "R. conorii complex" which includes the Indian tick typhus rickettsia, the Astrakhan fever rickettsia, and the Israeli spotted fever (ISF) rickettsia (6, 17, 19). Recently, the creation of four R. conorii subspecies has been proposed in order to separate these strains on the basis of genetic and serological methods (26). Therefore, rickettsial isolates exhibiting close genetic similarity to the R. conorii Malish type strain (ATCC VR-613) should be classified as Rickettsia conorii subsp. conorii, while three further subspecies, Rickettsia conorii subsp. indica, Rickettsia conorii subsp. caspia, and Rickettsia conorii subsp. israelensis, have been created to accommodate isolates genetically similar to the type strains of the Indian tick typhus rickettsia (ATC C VR-597), the Astrakhan fever rickettsia (A-167), and the ISF rickettsia (ISTTCDC1), respectively (26).
R. conorii subsp. israelensis was first isolated in 1974 in Israel, where its distribution initially appeared to be restricted (19), but more recently it has also been isolated in Portugal (3). In Italy, a number of different spotted-fever-group rickettsiae have been detected in Ixodes ricinus ticks from central and northern regions, raising the possibility that bacteria other than R. conorii are involved in rickettsial diseases in our country (4). Our recent finding of R. conorii subsp. israelensis infection in a Rhipicephalus sanguineus tick, which is the main vector for MSF in Sicily, also suggested that the geographic distribution of ISF might be wider than previously thought, including not only Israel and Portugal but also Italy (7). To check whether unusual tick-transmitted rickettsiae are actually involved in MSF in Sicily, we used molecular-sequence-based identification techniques to retrospectively study clinical isolates obtained from several cases of MSF over a 15-year period and previously identified by serological tests as belonging to the spotted-fever-group rickettsiae.
MATERIALS AND METHODS
Rickettsial isolates. Rickettsiae of the spotted fever group were isolated by the shell vial technique from blood samples collected from 24 patients hospitalized for MSF in Palermo, Sicily, Italy, in the years 1987 to 2001. Heavily infected Vero cell monolayers were harvested and stored at –80°C until use. R. conorii subsp. conorii type strain Malish (ATCC VR-613) was kindly provided by D. H. Walker and was used as a control strain.
PCR amplification. Bacterial DNA was obtained from 200 μl of infected Vero cell suspensions by using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI) according to the manufacturer's instructions. PCR was performed with the primer pair Rr 190.70p-190.701, which amplifies 632-bp portions of the ompA gene, under conditions described previously (18). Two microliters of the DNA preparation was amplified in a 100-μl reaction mixture containing 10 pmol each primer; 200 μM (each) dATP, dCTP, dGTP and dTTP (Amersham Pharmacia Biotech, Uppsala, Sweden), 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 6 μl of a 25 mM solution of MgCl2. Amplifications were carried out in a DNA thermal cycler (GeneAmp PCR System 2400; Perkin-Elmer, Applied Biosystems Division, Norwalk, CT) under the following conditions. An initial 3 min of denaturation at 95°C was followed by 35 cycles of denaturation for 20 s at 95°C, annealing for 30 s at 46°C, and extension for 1 min at 65°C. The amplification was completed by holding for 7 min at 72°C. PCR products were purified as previously described by Roux et al. (18).
Restriction analysis. Amplified products were submitted to restriction analysis with endonucleases PstI and RsaI (Amersham Pharmacia Biotech) as previously described (18). Restriction fragments were resolved on 10% polyacrylamide gels, and electrophoresis was performed in 1x TBE buffer, pH 8 (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA), at 120 V for 3 h. Gels were stained with ethidium bromide and examined with a UV transilluminator. HaeIII-digested pUC18 double-stranded DNA (Sigma-Aldrich, St. Louis, MO) was used as a molecular weight marker to calculate the sizes of the restriction fragments.
Sequence analysis. To confirm the presumptive identification obtained by restriction analysis, PCR products of the Israeli spotted fever rickettsia isolates were sequenced in both the sense and antisense orientations (MWG-Biotech, Germany), and forward and reverse sequence data of each sample were aligned using the Bioedit program to obtain the final sequence. The DNA sequences obtained in this study were analyzed using the BLAST program, available through the National Center for Biotechnology Information (NCBI, Bethesda, MD; http://www.ncbi.nlm.nih.gov/BLAST/) (1). The phylogenetic relationship between our sequences and those of other rickettsiae of the SFG, retrieved from the GenBank database and selected on the basis of previous reports (6), was studied. Complete alignment was performed with CLUSTAL W (21), and the alignment was analyzed using the Kimura 2-parameter model as a method of substitution and neighbor joining to reconstruct the phylogenetic tree, using MEGA 3.0 software (http://www.megasoftware.net/) (8). The statistical significance of the phylogenies constructed was estimated by bootstrap analysis with 1,000 pseudoreplicate data sets, and a tree was displayed with the MEGA program.
Serological diagnosis. Results of serological diagnosis were recovered from recorded data. Specific antirickettsial immunoglobulin M (IgM) and IgG determinations had been performed by an enzyme-linked immunosorbent assay (ELISA) or an indirect immunofluorescence (IIF) assay. In both cases, antigens had been obtained from a clinical isolate of R. conorii subsp. conorii (strain MAVI). Antigens had been freshly prepared in the hospital laboratory as described previously (23).
Statistical analysis. Epidemiological and clinical data of the two groups of patients, those infected by R. conorii subsp. conorii and those infected by R. conorii subsp. israelensis, were compared and submitted to statistical analysis by the 2 test or the F test for analysis of variance. Results were considered significant when the P value was 0.05.
Nucleotide sequence accession number. The ompA gene sequence of the Italian clinical isolates of R. conorii subsp. israelensis described in this study has been deposited in GenBank and assigned accession number AY197564.
RESULTS
All the infected Vero cell suspensions from our collection were positive for the ompA gene by PCR. Restriction analysis with endonucleases PstI and RsaI was performed on all the amplicons obtained. The observation of a peculiar 3-band PstI restriction profile allowed presumptive identification of five clinical isolates as belonging to R. conorii subsp. israelensis, while the remaining 19 rickettsial isolates were R. conorii subsp. conorii (Table 1). The oldest R. conorii subsp. israelensis strain was isolated in 1991, while the other four were obtained in 1995, 2000 (two), and 2001. Analysis of the sequences obtained from the amplified ompA fragments of the five clinical R. conorii subsp. israelensis isolates showed their complete identity. Comparison with homologous sequences from closely related SFG Rickettsia spp. retrieved from GenBank showed 100% identity to the reference R. conorii subsp. israelensis strain registered under accession number U43797, as well as to the rickettsia we isolated from a tick in 1990 (7). Figure 1 shows the results of the phylogenetic analysis performed on 596-bp partial sequences of the PCR-amplified ompA genes of the five human isolates, confirming their identification as R. conorii subsp. israelensis.
Epidemiological, clinical, and laboratory data for the five patients were recovered from the archives and are given in Tables 2 and 3.
Before the onset of the disease, none of the patients had a history of tick bite, tick exposure, or a stay in an epidemiologically suspected location. The illness had a sudden onset with fever, but no specific symptoms appeared during the first several days. In all the patients, a macular or maculopapular rash appeared. Only two patients had a tache noire (eschar). Initial antibacterial treatment at home (with oral penicillins or cephalosporins) had been started by three patients but did not result in improvement. Five to 8 days after the onset of symptoms, the patients were admitted to the hospital. Although they immediately received oral doxycycline therapy, in three cases disseminated intravascular coagulation (DIC) and progression toward coma was observed. One of these patients died the day after admission, and DIC with acute renal and hepatic failure was diagnosed on the basis of laboratory tests showing severe thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time, the presence of fibrin degradation products and D-dimer, elevated levels of serum transaminases, bilirubin, creatinine, and blood urea nitrogen, decreased creatinine clearance, hepatomegaly, and splenomegaly. No ultrasound test or liver biopsy was performed to ascertain the extent of hepatic necrosis. The other two coma patients recovered after 18 and 21 days, respectively. The remaining two ISF cases were milder, with clinical symptoms resolving after 7 and 12 days, respectively.
The serological data we recovered showed that specific antirickettsial Ig determinations had been performed for all of the patients. With the single exception of the patient who died the day after admission, at least two serum samples per patient had been obtained and tested during hospitalization. For four patients, serological tests showed evidence of acute rickettsial infection, e.g., the presence of IgM antibodies, seroconversion, or a fourfold rise in antibody titer against SFG rickettsial antigens (Table 3). In three cases, sera reacted at high titers with the R. conorii subsp. conorii MAVI strain used for these tests. The two serological tests used were generally in agreement, but the IgG titer (slightly increasing in paired samples) revealed by ELISA for patient 1 was not detected by IIF (Table 3).
The clinical data recovered for the 19 patients suffering from classical Rickettsia conorii subsp. conorii MSF showed that all but 2 had mild forms of disease and 12 had a typical eschar. The mean age of R. conorii subsp. conorii MSF patients was 54 (range, 26 to 74 years).
DISCUSSION
Our study demonstrates the presence of ISF caused by R. conorii subsp. israelensis in Sicily. This result was not surprising, since the infection had already been detected in R. sanguineus ticks, the main vector for MSF in Sicily (7). Moreover, we were able to trace back the infection in humans at least to the beginning of the 1990s, since the first reported case was in 1991. Therefore, we can now affirm that the distribution of ISF is wider than previously thought and includes not only Israel and the Iberian Peninsula but also Sicily.
The Rhipicephalus sanguineus tick is the main vector for MSF in Mediterranean countries. Although contact with ticks could not be documented from the recorded anamnestic data, it must be assumed also for Sicilian ISF patients and can be granted by the presence of the typical eschar associated with the tick bite site which was observed on two of them. The Sicilian ISF patients lived in urban or semirural areas of western Sicily, and none of them had left Italy during the previous year. R. conorii subsp. israelensis strains might have been imported from areas of endemicity through imported ticks (e.g., on migrating birds) or rodents in recent years, but a long history of endemicity in Sicily cannot be excluded. The seasonal distribution of infections was between June and September, corresponding to that generally observed for MSF in our region. Although the disease apparently affected slightly younger people than classical R. conorii subsp. conorii MSF (mean ages, 49 and 54 years, respectively), this difference was not statistically significant, and only one of five ISF patients was <49 years old.
Clinical diagnosis of MSF relies on the following symptoms: fever, tache noire, and rash. The absence of tache noire, the characteristic eschar at the site of the tick bite, has been described for Israeli spotted fever (3, 19). When the typical tache noire sign is not present, there is a risk of delay in administration of appropriate antibiotic treatment. The inconsistent presence of the tache noire had already been observed in the past for MSF patients in Sicily (24). In this study, only two out of five ISF patients had a typical MSF tache noire, but the absence of this feature was not significantly related to ISF patients. Although the recorded clinical data we found on the Sicilian ISF patients did not allow calculation of a precise severity score, three out of five patients could be considered to have suffered from severe forms of disease, since one died of his illness and for the remaining two patients impaired consciousness and progression toward coma was observed, and hospitalization in intensive care units, for more than 15 days, was required. Both the appearance of DIC signs and loss of consciousness were significantly associated with ISF (Table 2). The supposed ability of the Israeli spotted fever rickettsia to cause life-threatening disease has been ascribed to late diagnosis due to uncharacteristic presentation (25). An increase in severe cases of MSF has been seen in Sicily in recent years (11) and in Portugal in 1997, where it has been related to the presence of R. conorii subsp. israelensis (2, 5). A fulminant course of Israeli spotted fever has been correlated with glucose-6-phosphate dehydrogenase deficiency (16), but none of the Sicilian ISF patients had been tested for this.
The fact that a rapidly fatal outcome can occur stresses the importance of prompt diagnosis and treatment, even for apparently benign disease. In most cases, diagnosis of MSF is based on serology. The serological data of our patients showed that R. conorii subsp. israelensis produces a clear immunological response, providing evidence for acute rickettsial infection, and that serological tests using R. conorii subsp. conorii antigens are able to detect it, confirming that cross-reactions are common among rickettsiae of the two subspecies (26). However, in some cases antibodies could be detected only several days after the onset of the disease. Confirmatory diagnosis in the acute phase must rely either on the detection of rickettsiae in the clinical specimens by culture isolation with the shell vial technique, followed by identification using immunohistochemistry or the indirect fluorescent antibody test, or on the detection of rickettsial DNA by PCR. The PCR tests have been demonstrated to allow fast detection of as few as 100 copies of rickettsial DNA through amplification of specific sequences of the genes encoding the 16S rRNA, the 17-kDa protein, citrate synthase (gltA), and the outer membrane proteins OmpA and OmpB (9, 10). However, in early stages and until diagnosis continues to be clinical, patients should start appropriate therapy without delay if suspicion of rickettsiosis arises in order to prevent a poor outcome due to aggressive rickettsial strains. Prevention programs aimed at containing contact with infected ticks should be encouraged not only in Sicily but also in countries where rickettsial diseases are endemic.
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