| Abstract |
Extended summary
Historical Background: Rickettsiaceae family of microbes first appeared on earth 150 million years ago. Their common ancestors were Proteobacteria which had 3000- 5000 genes. During the evolutionary course, the common ancestor of the genus Rickettisiae shed a great part of the initial genome (almost 1254- 1700 genes) and lost the ability to compose amino acids and nucleotides. As a consequence the microorganism needed to live intracellularly as a parasite to the host organism. Initially, this co-existence did not have any consequences for the hosting cells, later on the parasitic organism started destroying the cells leading to disease. The evolutionary course was rapidly accelerating until 50 million years ago when the arthropod-borne rickettsial diseases constituted a separate branch of the rickettsial diseases. Rickettsial diseases made their presence especially evident during the Middle Ages when the epidemic typhus claimed more victims than the wars. In the end of the 19th and the beginning of the 20th century the first cases of ricketssial diseases were described in N. America. Military doctor Marshal Wood first described Rocky Mountain spotted fever (RMSF) in 1896. In 1907 Howard Ricketts described the first Rickettsia species that was named after his name but in year 1910 he died of the disease he described. In 1914 Prowazek described the presence of Rickettsia species in louse feces but he died of the disease shortly later. In 1928 Mooser confirmed Neil’s research by inoculating microorganisms from a patient’s serum to guinea pigs and he contributed to the distinction between epidemic and endemic typhus. In 1930 Dr. Joseph Lipsky got sick with fever and rash and he tested positive for Weil-Felix test. It was then that rats and fleas were confirmed as carriers of endemic typhus. In 1932 Lepine confirmed that typhus cases in Athens were associated with rats from which he isolated Rickettsiae organisms (then called Ratvirus). In 1932 Blanc and Kaminopetros proved that the tick R. sanguineus was the transmitter of R.conorii, the causing organism of Mediterranean spotted fever. In 1970 David Walter proved the presence of R.ricketsii in skin rash of a patient suffering of RMSF. In 1981 Philip isolated Rickettsiae organisms in cell cultures using immunofluorescence. With the progress of molecular biology, in 1998 the genome of R. prowazekii was confirmed and later on in 2002 the genome of R.conorii. In the 1980s the research team of I. Tselentis described 49 cases of endemic typhus in the island of Evia, Greece.
Taxonomy: The exclusively intracellular growth of the rickettsiae led to significant taxonomy implications. Rickettsiae are described as small, intracellular Gram-negative bacilli that can be stained with Gimenez stain and their growth is totally dependent on their presence in eukaryotic cells. In 1974 the Chlamidiacae family was separated from the Ricketssials class. After several sub classifications the final taxonomy in Rickettsiae, Coxiella and Rochalimea based on their culture characteristics and their incubation in eukaryotic cells was proposed. Eventually Rickettsiae genus was divided in three groups:
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The Spotted Fever Group, the Typhus Group and the Scrub Typhus Group. In the 1990s the molecular techniques based basically on the 16sRNA sequence change the taxonomy once again. The combination of different genome sequences led to admirable phylogenetic diagrams. The latest Rickettsiae classification was based on the Sca4 gene and it consists of the following five groups: 1. The Typhus group 2. The R.ricketsii group 3. The R.massiliae group 4. The R.helvetica group and 5. The R.conorii, R. acarii, R. felis, and R.australis group. This classification is also consistent with the analyses based on Sca1 and Sca2 genes. Their cellular structure with the 3-layer cell wall gave researchers the opportunity to develop diagnostic techniques like the Weil-Felix serodiagnostic procedure which is based on the cross reaction between the rickettsial antigens and the ones of Proteus vulgaris (OX19 for the Typhus group and OT and OX2 for the spotted fevers group). This method detects IgG antibodies. Later on, more diagnostic methods developed, like the complement fixation method (CF), the indirect hemoagglutination, ELISA, the indirect immunofluorescence (IFA) and Western Blot. Molecular biology contributed to the diagnosis of the disease with PCRrflp sequencing.
In conclusion, Rickettsiae are fastidious bacterial organisms that are obligate intracellular parasites. They are small pleomorphic coccobacilli. Their bacterial nature is well established; they multiply by binary fusion, contain both RNA and DNA and have both synthetic and energy-producing enzyme systems.
Pathophysiology of rickettsial diseases: The pathogenic rickettsias are transmitted to humans via arthropods like ticks, lice and fleas who parasitize on the vertebrate hosts. Humans are accidental hosts who presumably become infected when infected flea feces contaminate a pruritic flea bite wound or the conjunctivae of the host. After their entrance in the organism through capillaries or lymph vessels, the rickettsias settle into the endothelial cells where they multiply and they disseminate in all the host organs and they cause disease. The involvement of the endothelial cells causes vasculitis which can explain the clinical manifestations of the disease (rash, central nervous system, lung, kidney and heart involvement). Rickettsias entry into the skin activates the dendritic cells which migrate to the lymph nodes and the immune mechanisms are activated. The recent decoding of the genome clarified the pathogenicity mechanisms. The rickettsiae of the spotted fever group grow in both nucleus and cytoplasma whereas the rickettsiae of the Typhus group grow only in the cytoplasma. Endothelial involvement causes edema, destruction and cellular apoptosis which results to epithelial denudement, and coagulation mechanism activation. These manifestations account for the hema tological complications noted such as thrombopenia.
Immunity: The rickettsial entrance into the cell activates NFkB which promotes the IL6-IL19 production as well as IL18 and MCP-1. Eventually and through the CD8 T-lymphocyte preponderance, apoptosis of
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the endothelial cells occurs. The P38MAP kinase leads to the secretion of a variety of inflammatory and chemotactic cytokines. TNF-alpha and INF-γ promote the iNOS production which is lethal for the rickettsiae. Eventually, the intracellular rickettsiae are killed through H2O2 production from the macrophages and NO production from the RANTES liver cells. Among the rickettsiae, there are specific small differences regarding the cytokine expression in the infected macrophages.
Acquired Immunity: The dendritic cells of the skin present the antigens to CD4 helper lymphocytes and lead to CD8 activation, the lymphocytes that kill the infected cells. Antibodies against rOmpA and rOmpB antigens protect mice from lethal infection. Rickettsiae eventually are destroyed through NO as well as L-Tryptophan deprivation which is necessary for the rickettsial metabolism. Humoral immunity disorders may lead to the production of the anti-inflammatory cytokine IL-10 which stimulates Treg and suppressor lymphocytes resulting to the uncontrolled rickettsial growth and bad disease outcome.
Acute phase reaction: During the acute phase there is an increase of INF-γ, IL6, TNF-alpha, IL10 but IL1 and IL8 are non-detectable. C3, C4, CRP and factor B increase during the first two weeks of the disease and consequently they return to normal levels. Fibrinogen increases during the first week of the disease. In a lethal case of spotted fever disease in Northern Greece the TNF levels were undetectable.
Treatment: Doxycycline is the treatment of choice with the MICs of the pathogens ranging between 0.06 and 0.25 μg/ml according to the literature. Chloramphenicol is equally effective but it is not the drug of choice because of the well known side effects. Ketolide telithromycin is effective, as well as macrolides such as erythromycin, clarithromycin, azithromycin and roxithromycin. Josamycin is a newer active antibiotic but more experience is needed. The fluoroquinolones levofloxacin and ofloxacin are active against R. conorii and R. prowasekii but several reports regarding lack of effectiveness make their use still questionable. Rickettsiae are resistant to beta-lactams, aminoglycosides and cotrimoxazole. Recent research indicates that statins may protect against R. conorii disease.
Vaccines: The attempts for the development of a vaccine have not yielded any encouraging results.
Biological Warfare: R. prowazekii, R. typhi and R. ricketsii have been used in the form of aerosolized inhaler. The disease attack rate is 100% and mortality rate ranges from 10% to 60%. Because of this significant risk, CDC categorizes R.prowazekii as Biological Weapon Risk Grade B and the rest of the Rickettsiae as Biological Weapons Risk Grade C. Their use has been dated back to 1930s and 1940s in China and Japan.
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PRESENT RESEARCH:
The re-emergence of endemic typhus in the area of Chalkida, Evia (Central Greece) was the cause for this study.
Materials and Methods: 174 patients who presented to the General Hospital of Chalkida with the clinical syndrome of murine typhus (MT) between 1999- 2005 were included. The minimum presumptive clinical criteria for MT included fever, rash and headache and the absence of any other clinically identified site of infection. Three blood samples were obtained: upon admission, a week and three weeks later. The basic laboratory tests were obtained upon admission. White blood cell buffy coat was preserved in -80° C in order for further work to be performed in the Laboratory of Clinical Bacteriology, Parasitology and Zoonoses and Geographical Medicine in the University of Crete. A complete epidemiologic history of the patients was documented. In some cases this process included visits at residence and work environments. Indirect immunofluorescence serology was performed for R. typhi as well as other rickettsial types. Cultures from the white blood cells buffy coats were performed in shel vials for pathogen isolation. PCR was performed in the whole blood samples. IgG titers of ≥1:960 or IgM titers ≥1:400 and/or a 4-fold increase in titers between two successive assays were considered as indicative of acute infection. Fifty four rats were collected from the patients’ environments and blood samples were also collected from them. Thirty six X. cheopis fleas were collected from the rats and 23 C. felis fleas from the patients’ environment.
Results: The clinical syndrome was described in 90 patients, as well as a full recording of the clinical course and the laboratory and epidemiologic data. Among the rat samples, five were tested positive for serology to R. typhi. Three among 36 X. cheopis fleas were tested positive for R. typhi and two among 23 C. felis fleas were tested positive for R. felis. Three patients were tested positive to R. felis and one to R.mongoltimone. C.burnetti was isolated from the buffy coat of six patients and were excluded from the study. Among the 174 patients, 128 were studied. Full analysis was performed for a total of 90 adults and 20 geriatric patients. The study of these two groups of patients reveals that endemic typhus is still prevalent in the area. The classic clinical triad includes fever, headache and maculopapular rash. Main laboratory findings include transaminasemia, anemia, hypoalbuminemia and hyponatremia. Therapeutic response to doxycycline was successful with a shorter interval to defervescence compared to ofloxacin. Complications included lung infiltrates, pleurisy, renal failure, coma and lethargy and were almost 3-fold more in the geriatric subpopulation. Therapeutic response was successful in both teams, mainly because of the high index of suspicion for the disease. The presence of two new rickettsial species imposes increased vigilance for further investigation.
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Conclusions: Rickettsioses are more common than most physicians suspect. The diseases are probably under-diagnosed and largely under-reported. Treatment is simple and may avert the serious and occasionally fatal complications. Increased awareness of the disease is necessary in endemic areas for early administration of effective treatment.
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