76 Am. J. Trop. Med. Hyg., 66(1), 2002, pp. 76–79 Copyright 2002 by The American Society of Tropical Medicine and Hygiene PREVALENCE OF RICKETTSIA CONORII AND RICKETTSIA TYPHI INFECTIONS IN THE POPULATION OF NORTHERN GREECE S. ALEXIOU DANIEL, K. MANIKA, M. ARVANITIDOU, AND A. ANTONIADIS A’ Department of Microbiology, School of Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece; Laboratory of Hygiene, School of Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece Abstract. Seroepidemical surveys concerning the prevalence of Rickettsia conorii and Rickettsia typhi have never been studied in northern Greece. We examined 1,584 sera samples from residents of northern Greece for the detection of antibodies to R. conorii and to R. typhi by means of immunofluorescence assay. In addition, we compared the prevalence of rickettsial infections among the demographic variables of sex, age, occupation, and area of residence. Antibodies to R. conorii and R. typhi were found in 125 (7.9%) and 31 (2.0%) of the examined subjects, respectively. The prevalence of antibodies to R. conorii correlated with increasing age and was statistically higher in men. Farmers had significantly higher prevalence of antibodies to both species of rickettsiae studied versus other professions. Residents of rural areas showed a statistically higher prevalence for R. conorii versus urban residents, although this difference was not demonstrated for R. typhi. We also detected differences in the prevalence of rickettsial infections among the different prefectures. Our data show the wide distribution of R. conorii in northern Greece and indicate the presence of R. typhi. INTRODUCTION The Rickettsiae is a group of bacteria that causes disease in both animals and humans. The cause of Mediterranean spotted fever is Rickettsia conorii, and it is reported to be transmitted to humans by the bite of the dog tick Rhipiceph- alus sanguineus. 1 Rickettsia typhi (mooseri) is the cause of murine or endemic typhus and is transmitted to humans by the bite of the rat flea Xenopsylla cheopis. 2 The clinical man- ifestations of rickettsioses vary from mild symptoms that often do not require hospitalization to severe illness that can result in death. 3 Rickettsiosis is not a reportable disease in Greece; hence, its significance is often underestimated. The prevalence of rickettsioses in the population of north- ern Greece has not been studied to date. This region was selected because of its importance as a neighbor area to Al- bania, the former Yugoslav republic of Macedonia, and Bul- garia. The results of this serosurvey might also be indicative of the situation in these countries. The purpose of this study was to evaluate the prevalence of rickettsial infections in the population of northern Greece and to determine its distri- bution among several demographic variables, including sex, age, occupation, and area of residence. MATERIALS AND METHODS Geographical area. The area of northern Greece studied in this survey consisted of 14 prefectures measuring 37,542 km 2 . The topography is divided into plains (35%), mountains (39%), and hills (26%).The climate is mild near the coast- line, and it is continental inland. The total population of the area studied was 2,256,160 inhabitants. The most common occupations were farming, livestock breeding, or both (32%) and industry (22%). Samples. Between April and October of 2000, sera sam- ples from 1,584 randomly selected people (567 men and 1,017 women with mean age standard deviation of 54.4 18.6 years) were collected by the prefecture hospitals of northern Greece and sent to our laboratory. The collection of the sera took place during a 2-week period in each hos- pital. The subjects showed no evidence of febrile illness, had no known history of rickettsiosis, and presented as outpa- tients for routine biochemical tests. Consent was obtained from each subject that the serum could be used for the de- tection of antibodies against several infectious agents. The study was approved by the ethical committee of AHEPA hospital, University of Thessaloniki. The following infor- mation was recorded for each person: age, sex, occupation, and area of residence. The study population was stratified by age in groups of 0–14 years, 15–29 years, 30–44 years, 45–64 years, and 65 years, and by demographic area— that is, rural (10,000 inhabitants) and urban (10,000 inhabitants). Serological technique. Serum samples were tested by in- direct immunofluorescence with commercially available an- tigens (Rickettsia conorii Spot IF, Rickettsia mooseri Spot IF, BioMerieux, Marcy l’ Etoile, France). The antigens con- tained Rickettsia typhi and Rickettsia conorii obtained from cultures on Vero cells and were fixed on the slides. All sam- ples were tested in a 1/64 initial dilution. After incubation for 30 min at 37C in a moist chamber, the slides were washed 3 times with phosphate-buffered saline. Afterward, we added anti-human immunoglobulin (Ig) G sheep globulin labeled with fluorescein (Sanofi Diagnostics, Pasteur) in a 1/20 titer, as determined by chessboard titration, containing Evans blue. The slides were incubated at 37C for 30 min in a moist chamber and washed again as previously de- scribed. Negative and positive sera samples from our labo- ratory collection were used in each test as negative and pos- itive controls. Intense fluorescence of the Rickettsiae situated in or outside the cells was considered to be a positive re- action. In negative reactions, the cells were tinted red and did not display any fluorescence. End-point titers were ob- tained by serial dilution on positive specimens. Titers of 1/64 or higher were considered positive. Statistical analysis. Biostatistical analysis was performed by the statistical package SPSS, version 10.0.1 (Standard version, for Windows; SPSS, Chicago, IL). Student’s t-test was used for comparing the mean ages between seropositive and seronegative subjects against R. conorii and R. typhi. The chi-square test (with or without Yate’s continuity cor- rection) and Fisher’s exact method for small samples were
Transcript
Monday Jan 14 2002 02:27 PM 2000Allen Press • DTPro System
tmed 66_117 Mp_76File # 17tq
76
Am. J. Trop. Med. Hyg., 66(1), 2002, pp. 76–79Copyright � 2002 by The American Society of Tropical Medicine and Hygiene
PREVALENCE OF RICKETTSIA CONORII AND RICKETTSIA TYPHI INFECTIONS INTHE POPULATION OF NORTHERN GREECE
S. ALEXIOU DANIEL, K. MANIKA, M. ARVANITIDOU, AND A. ANTONIADISA’ Department of Microbiology, School of Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece;
Laboratory of Hygiene, School of Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece
Abstract. Seroepidemical surveys concerning the prevalence of Rickettsia conorii and Rickettsia typhi have neverbeen studied in northern Greece. We examined 1,584 sera samples from residents of northern Greece for the detectionof antibodies to R. conorii and to R. typhi by means of immunofluorescence assay. In addition, we compared theprevalence of rickettsial infections among the demographic variables of sex, age, occupation, and area of residence.Antibodies to R. conorii and R. typhi were found in 125 (7.9%) and 31 (2.0%) of the examined subjects, respectively.The prevalence of antibodies to R. conorii correlated with increasing age and was statistically higher in men. Farmershad significantly higher prevalence of antibodies to both species of rickettsiae studied versus other professions.Residents of rural areas showed a statistically higher prevalence for R. conorii versus urban residents, although thisdifference was not demonstrated for R. typhi. We also detected differences in the prevalence of rickettsial infectionsamong the different prefectures. Our data show the wide distribution of R. conorii in northern Greece and indicatethe presence of R. typhi.
INTRODUCTION
The Rickettsiae is a group of bacteria that causes diseasein both animals and humans. The cause of Mediterraneanspotted fever is Rickettsia conorii, and it is reported to betransmitted to humans by the bite of the dog tick Rhipiceph-alus sanguineus.1 Rickettsia typhi (mooseri) is the cause ofmurine or endemic typhus and is transmitted to humans bythe bite of the rat flea Xenopsylla cheopis.2 The clinical man-ifestations of rickettsioses vary from mild symptoms thatoften do not require hospitalization to severe illness that canresult in death.3 Rickettsiosis is not a reportable disease inGreece; hence, its significance is often underestimated.
The prevalence of rickettsioses in the population of north-ern Greece has not been studied to date. This region wasselected because of its importance as a neighbor area to Al-bania, the former Yugoslav republic of Macedonia, and Bul-garia. The results of this serosurvey might also be indicativeof the situation in these countries. The purpose of this studywas to evaluate the prevalence of rickettsial infections in thepopulation of northern Greece and to determine its distri-bution among several demographic variables, including sex,age, occupation, and area of residence.
MATERIALS AND METHODS
Geographical area. The area of northern Greece studiedin this survey consisted of 14 prefectures measuring 37,542km2. The topography is divided into plains (35%), mountains(39%), and hills (26%).The climate is mild near the coast-line, and it is continental inland. The total population of thearea studied was 2,256,160 inhabitants. The most commonoccupations were farming, livestock breeding, or both (32%)and industry (22%).
Samples. Between April and October of 2000, sera sam-ples from 1,584 randomly selected people (567 men and1,017 women with mean age � standard deviation of 54.4� 18.6 years) were collected by the prefecture hospitals ofnorthern Greece and sent to our laboratory. The collectionof the sera took place during a 2-week period in each hos-pital. The subjects showed no evidence of febrile illness, had
no known history of rickettsiosis, and presented as outpa-tients for routine biochemical tests. Consent was obtainedfrom each subject that the serum could be used for the de-tection of antibodies against several infectious agents. Thestudy was approved by the ethical committee of AHEPAhospital, University of Thessaloniki. The following infor-mation was recorded for each person: age, sex, occupation,and area of residence. The study population was stratifiedby age in groups of 0–14 years, 15–29 years, 30–44 years,45–64 years, and � 65 years, and by demographic area—that is, rural (� 10,000 inhabitants) and urban (� 10,000inhabitants).
Serological technique. Serum samples were tested by in-direct immunofluorescence with commercially available an-tigens (Rickettsia conorii Spot IF, Rickettsia mooseri Spot IF,BioMerieux, Marcy l’ Etoile, France). The antigens con-tained Rickettsia typhi and Rickettsia conorii obtained fromcultures on Vero cells and were fixed on the slides. All sam-ples were tested in a 1/64 initial dilution. After incubationfor 30 min at 37�C in a moist chamber, the slides werewashed 3 times with phosphate-buffered saline. Afterward,we added anti-human immunoglobulin (Ig) G sheep globulinlabeled with fluorescein (Sanofi Diagnostics, Pasteur) in a1/20 titer, as determined by chessboard titration, containingEvans blue. The slides were incubated at 37�C for 30 minin a moist chamber and washed again as previously de-scribed. Negative and positive sera samples from our labo-ratory collection were used in each test as negative and pos-itive controls. Intense fluorescence of the Rickettsiae situatedin or outside the cells was considered to be a positive re-action. In negative reactions, the cells were tinted red anddid not display any fluorescence. End-point titers were ob-tained by serial dilution on positive specimens. Titers of1/64 or higher were considered positive.
Statistical analysis. Biostatistical analysis was performedby the statistical package SPSS, version 10.0.1 (Standardversion, for Windows; SPSS, Chicago, IL). Student’s t-testwas used for comparing the mean ages between seropositiveand seronegative subjects against R. conorii and R. typhi.The chi-square test (with or without Yate’s continuity cor-rection) and Fisher’s exact method for small samples were
77RICKETTSIA CONORII AND RICKETTSIA TYPHI IN GREECE
TABLE 1Antibodies to Rickettsia conorii and Rickettsia typhi according to
age group
Age
Rickettsia conorii
Negative,n
Positive, n(%)
Rickettsia typhi
Negative,n
Positive, n(%) Total
0–1415–2930–4445–64�65Total
22180228490539
1,459
1 (4.35)9 (4.76)
14 (5.78)37 (7.02)64 (10.6)
125 (7.89)
23188239517586
1,553
01 (0.52)3 (1.23)
10 (1.89)17 (2.81)31 (1.96)
23189242527603
1,584
TABLE 2Prevalence of antibodies to Rickettsia conorii and Richettsia typhi
FIGURE 1. Map of northern Greece indicating the prevalence of antibodies to Rickettsia conorii in each prefecture.
used for the comparison of the prevalence rates of the sub-group analysis. The chi-square Mantel-Haenszel procedureevaluated trends for categorical variables.
RESULTS
Of the 1,584 total patients, antibodies to R. conorii werefound in 125 (7.9%); antibodies to R. typhi were found in31 (2.0%). The age of the seropositive people to R. conoriiwas (mean age � standard deviation) 58.97 � 16.97, where-as seronegative people had an age of 54.03 � 18.64 (P �0.05). The mean age (61.65 � 12.75) of the R. typhi–sero-positive population was also statistically higher than themean age (54.27 � 18.63) of the seronegative population (P� 0.001). The highest prevalence of antibodies to R. conoriiwas observed in the � 65-year-old age group, with a trendof increasing prevalence with age. Differences of prevalencebetween age groups reached statistical significance (P �0.05). The same trend was observed for R. typhi, although
differences between groups were not statistically significant(Table 1). Fifty-nine (10.4%) of 567 men and 66 (6.5%) of1,017 women expressed antibodies against R. conorii (P �0.005), whereas the prevalence of antibodies against R. typhiwas 1.9 and 2.0%, respectively, for the 2 sexes.
The prevalence of antibodies was statistically different inthe 14 prefectures. This varied from 1.0 to 20.0% for R.conorii (P � 0.001) and 0–7.0% for R. typhi (P � 0.05;Table 2; Figures 1 and 2). Farmers and livestock breedershad significantly higher prevalences of antibodies comparedwith other professions for both R. conorii (P � 0.001) andR. typhi (P � 0.01; Table 3). Inhabitants of rural areas
78 ALEXIOU DANIEL AND OTHERS
FIGURE 2. Map of northern Greece indicating the prevalence of antibodies to Rickettsia typhi in each prefecture.
TABLE 3Prevalence of antibodies according to profession and area of residence
Variable
Positive for Rickettsia conorii
n % (P value)
Positive for Rickettsia typhi
n % (P value) Total
FarmersOther professionsRuralUrban
9827
12130
10.24.3 (�0.001)
10.66.8 (�0.05)
265
2110
2.70.8 (�0.01)
1.82.3 (NS)
959625
1,146434
NS � not statistically significant.
showed a higher prevalence of antibodies to R conorii versusurban inhabitants (10.6 and 6.8%, respectively; P � 0.05);however, this did not achieve statistical differences for R.typhi (1.8% versus 2.3%; Table 3).
DISCUSSION
During the past 2 years, 8 cases of R. conorii and 2 casesof R. typhi infection were diagnosed by our laboratory. Inmost of them, rickettsiosis was not suspected clinically.Blood samples were sent to the laboratory in order to beexamined for other infectious agents when the correct di-agnosis was made. We can therefore assume that rickettsiosisis often underdiagnosed.
Our results showed that 7.9% of the population of north-ern Greece are IgG positive to R. conorii, whereas the per-centage of the IgG-positive population to R. typhi is defi-nitely lower (2%). This observation is not surprising in thepopulation we studied because R. conorii is endemic in theMediterranean countries. In fact, other Mediterranean coun-tries have reported similar prevalence rates to those dem-onstrated in this study. In Catalonia, Spain, 8% of the pop-
ulation were reported to have antibodies to R. conorii.4 Insouthern France, 18% of 325 blood donors had antibodiesto R. conorii.5 Moreover, similar observations were made insoutheast Europe, such as in Croatia and in Bosnia and Her-zegovina.6,7
As for Greece, rickettsiosis is thought to be one of thepossible causes of the Athenian epidemic of 430–426 BC,which took place at the outbreak of the Peloponnesian Warand caused the death of Pericles.8 In recent years, seroepi-demiological surveys conducted in the island of Crete(southern Greece),9 the province of Fokida (centralGreece),10 and the province of Evia (central Greece)11–13 re-vealed the presence of antibodies to R. conorii and R. typhiin the population.
In contrast to other studies,4,14 the prevalence of antibodiesto both Rickettsiae that we studied increased with older peo-ple, and this correlation was statistically significant for R.conorii. This observation could be attributed to the differentlife conditions that existed some decades ago in this areaand to the longer period of exposure time experienced bythe older people.
Men appear to have antibodies to R. conorii more often
79RICKETTSIA CONORII AND RICKETTSIA TYPHI IN GREECE
Monday Jan 14 2002 02:27 PM 2000Allen Press • DTPro System File # 17tq
than women. This might be explained by the fact that menare more often occupied outdoor activities. However, no sig-nificant differences were found between the 2 sexes withantibodies to R. typhi. This observation can be attributed tothe fact that the flea-rat cycle, which is responsible for R.typhi infection, is often peridomestic.
The titers detected in this study were mostly low (1/64 to1/256). This finding is in agreement with other serosur-veys.4,14 Cases of cross reactions between R. conorii and R.typhi have been reported in the literature and were also de-tected in our study. Newhouse and others15 claim that thisfinding is less common with indirect immunofluorescencethan with other serological methods.
The highest percentages of seropositive people for bothR. conorii and R. typhi were observed among farmers. Thisfinding is in agreement with that of other surveys14 and canbe attributed to more frequent exposure. Moreover, the per-centage of people with antibodies to R. conorii who lived inrural areas was statistically higher than urban residents, re-gardless of their profession. This observation could be ex-plained by the fact that in rural areas, people are often in-volved in farming, even though their main profession mightbe different. However, where antibodies to R. typhi wereconcerned, there were no significant differences between res-idents of rural and urban areas. The peridomestic flea-ratcycle could be the reason for this observation.
The percentage of antibodies to R. conorii and R. typhivaried significantly among the prefectures studied. The high-est percentage of antibodies to R. conorii was detected inthe prefecture of Trikala (20% of the population studied inthis area), followed by Grevena (14%), Serrai (14%), andKastoria (10%). The highest percentage of antibodies to R.typhi was found in the prefecture of Khalkidiki (7%); all theother prefectures demonstrated a very low prevalence. Noenvironmental factors could explain this finding, as the an-nual mean temperature and precipitation is about the samein all the examined areas. Further research is being per-formed in this direction in collaboration with the regionalhospitals in order to investigate acute rickettsiosis. Epide-miological studies are also being done in order to isolate andidentify Rickettsiae in these high-risk areas.
Our data indicate that areas endemic for both Rickettsiaeexist in northern Greece. From the high prevalence of anti-bodies in northern Greece detected in this study, as in otherparts of the country,9–11 it is obvious that Rickettsiae arewidely distributed in Greece. This finding confirms the factthat rickettsioses remain a significant health problem inmany parts of the world.16
Acknowledgments: We thank the directors of the laboratories of theprefecture hospitals of northern Greece for collecting and sendingthe serum samples.
Authors’ addresses: S. Alexiou Daniel, K. Manika, and A. Anton-iadis, A’ Department of Microbiology, School of Medicine, Aristo-telian University of Thessaloniki 54006, Greece. M. Arvanitidou,Laboratory of Hygiene, School of Medicine, Aristotelian Universityof Thessaloniki 54006, Greece.
Reprint requests: S. Alexiou Daniel, A’ Department of Microbiology,School of Medicine, Aristotelian University of Thessaloniki, 54006,Greece, Telephone: �3031999159, Fax: �3031999149, E-mail:[email protected].
2. Farhahg-Azad A, Traub R, 1985. Transmission of murine typhusrickettsiae by Xenopsylla cheopis, with notes on experimentalinfection and effects of temperature. Am J Trop Med Hyg 34:555–563.
4. Segura-Porta F, Diestre-Ortin G, Ortuno-Romero A, Sanfeliu-Sala I, Font-Creus B, Munoz-Espin T, Mateu de Antonio E,Casal-Fabrega J, 1998. Prevalence of antibodies to spottedfever group rickettsiae in human beings and dogs from anendemic area of mediterranean spotted fever in Catalonia,Spain. Eur J Epidemiol 14: 395–398.
5. Raoult D, Toga B, Chaunet H, Chiche-Portiche C, 1987. Rick-ettsial antibodies in southern France: antibodies to Rickettsiaconorii, Coxiella burneti among urban, suburban and semi-rural blood donors. Trans R Soc Trop Med Hyg 81: 80–81.
6. Radulovic S, Walker DH, Weiss K, Dzelalija B, Morovic M1993. Prevalence of antibodies to spotted fever group rickett-siae along the eastern coast of the Adriatic sea. J Clin Micro-biol 31: 2225–2227.
7. Punda-Polic V, Leko-Grbic J, Radulovic S, 1995. Prevalence ofantibodies to rickettsiae in the northwestern part of Bosniaand Herzegovina. Eur J Epidemiol 11: 697–699.
8. Retrief FP, Cilliers L, 1998. The epidemic of Athens, 430–426BC. S Afr Med J 88: 50–53.
9. Antoniou M, Tselentis Y, Babalis T, Gikas A, Stratigakis N,Vlachonikolis I, Kafatos A, Fioretos M, 1995. The seroprev-alence of ten zoonoses in two villages of Crete, Greece. EurJ Epidemiol 11: 415–423.
10. Babalis T, Dupont HT, Tselentis Y, Chatzichristodoulou C,Raoult D, 1993. Rickettsia conorii in Greece: comparison ofa microimmunofluorescence assay and western blotting forseroepidemiology. Am J Trop Med Hyg 48: 784–792.
11. Tselentis Y, Babalis TL, Chrysanthis D, Gikas A, Chaliotis G,Raoult D, 1992. Clinicoepidemiological study of murine ty-phus on the Greek island of Evia. Eur J Epidemiol 8: 268–272.
12. Chniotis B, Psarulaki A, Chaliotis G, Gozalo Garcia G, Goza-dinos T, Tselentis Y, 1994. Transmission cycle of murine ty-phus in Greece. Ann Trop Med Parasitol 88: 645–647.
13. Tselentis Y, Psarulaki A, Maniatis J, Spyridaki I, Babalis T,1996. Genotypic identification of murine typhus rickettsia inrats and their fleas in an endemic area of Greece by the poly-merase chain reaction and restriction fragment length poly-morphism. Am J Trop Med Hyg 54: 413–417.
14. Ruiz-Beltran R, Herrero-Herrero JI, Martin-Sanchez AM, Mar-tin-Gonzalez JA, 1990. Prevalence of antibodies to Rickettsiaconorii, Coxiella burneti and Rickettsia typhi in SalamancaProvince (Spain). Serosurvey in the human population. Eur JEpidemiol 6: 293–299.
15. Newhouse VF, Shepard CC, Redus MD, Tzianabos T, McDadeJE, 1979. A comparison of the complement fixation, indirectfluorescent antibody and microagglutination tests for the se-rological diagnosis of rickettsial diseases. Am J Trop Med Hyg28: 387–395.
16. Raoult D, Roux V, 1997. Rickettsioses as paradigms of new oremerging infectious diseases. Clin Microbiol Rev 4: 694–719.
