- Email: [email protected]
Chlamydia pneumoniae IgG serological status and venous thromboembolism
Presse Med 2004; 33: 1493-6
L. de Saint Martin1, 1 2 E. Pasquier , F. Betsou , 3 1 A.Tran , F. Couturaud , 2 J. Orfila
1 - Internal Medicine and Chest Disease Department, University Hospital of Brest, France 2 - Biobanque of Picardie, Amiens, France 3 - Department of Microbiology, University Hospital of Brest, France Oral communication: 40th Interscience Conference on Antimicrobial Agents and Chemotheray, Sept 2000, Toronto, Abstract: 1985.
Corresponding author: L. de Saint Martin, Hôpital de la Cavale
A
© 2004, Masson, Paris
R T I C L E
O R I G I N A L
Chlamydia pneumoniae IgG serological status and venous thromboembolism A cross-sectional hospital based study
Summary
Résumé
Objective To search for a link between Chlamydia pneumoniae serological status and venous thromboembolic disease. Methods From March 1992 to October 1999, we conducted a crosssectional hospital-based study of consecutive unselected outpatients referred to us for clinical suspicion of venous thromboembolism. We compared the Chlamydia pneumoniae serological status with respectively, the venous thromboembolism, the deep vein thrombosis and the proximal deep vein thrombosis status. Results Among 1193 patients registered for suspected venous thromboembolism, 1010 samples were available (499 negative and 511 positive patients for venous thromboembolism). Seventy-nine patients were Chlamydia pneumoniae positive. Our work failed to demonstrate any clear association between Chlamydia pneumoniae and venous thromboembolism status. Nevertheless, we identified a statistical difference regarding Chlamydia pneumoniae seropositivity and proximal vein thrombosis status (adjusted odds ratio of 1.70, CI95%: 1.05 to 2.77). Conclusion The presence of Chlamydia pneumoniae antibodies might be a minor risk factor for venous thrombosis.
Statut sérologique IgG à l’égard de Chlamydia pneumoniae et maladie veineuse thromboembolique
L. de Saint Martin, E. Pasquier, F. Betsou, A. Tran, F. Couturaud, J. Orfila Presse Med 2004; 33: 1493-6 © 2004, Masson, Paris
Blanche, 29609 Brest cedex, France Tel: 02 98 34 73 36 Fax: 02 98 34 79 44 [email protected]
Étude d’un échantillon de patients hospitalisés Objectif Chercher un lien entre statut sérologique à l’égard de Chlamydia pneumoniae et maladie veineuse thromboembolique. Méthodes De mars 1992 à octobre 1999 tous les patients consécutifs hospitalisés pour suspicion clinique de maladie veineuse thromboembolique ont été inclus. Leur statut sérologique à l’égard de Chlamydia pneumoniae a été comparé respectivement au statut veineux thromboembolique, à l’existence d’une thrombose veineuse profonde et à celle d’une thrombose veineuse proximale. Résultats 1193 patients ont été admis pour suspicion de maladie veineuse thromboembolique ; 1 010 échantillons ont été disponibles (499 patients n’ayant pas de maladies veineuse thromboembolique et 511 en étant atteints). 79 patients avaient une sérologie positive pour Chlamydia pneumoniae. Il n’a pas été montré d’association entre le statut pour C. pneumoniae et l’état thromboembolique veineux. Néanmoins, il a été trouvé une différence statistique concernant la séropositivité pour C. pneumoniae et l’existence d’une thrombose veineuse proximale (odds ratio ajusté 1,70, intervalle de confiance 95 % : 1,05 à 2,77). Conclusion La présence d’anticorps à l’égard de Chlamydia pneumoniae peut être un facteur de risque minime de thrombose veineuse.
Received February, 5, 2003 Accepted April, 8, 2004
A
large number of seroepidemiological studies1, 2-4 confirmed by other data , have suggested an association between chronic Chlamydia pneumoniae (CP) infection and atherosclerosis. Several mechanisms of CP action have been suggested: endothelial injury,antigenic mimicry,increase of standard risk factors of atherosclerosis (dyslipidemia, body mass index), and/or procoagulant effects (dysfunction of pla1,5,6 telets or coagulation proteins) . In the event of coagulation protein dysfunction, a link between CP and venous thromboembolism (VTE) may be conceivable. Because the two previous seroepide7,8 miological studies gave conflicting results , we explored CP status in VTE patients.
4 décembre 2004 • tome 33 • n°21
Methods PARTICIPANTS From March 1992 to October 1999, all consecutive patients with a clinically suspected VTE admitted to the Internal Medicine and Chest Disease Department (University Hospital of Brest, Brittany, EU) were included.
CLINICAL AND BIOLOGICAL DATA All patients underwent a complete clinical work-up concerning well-known risk factors for VTE (prolonged immobilisation >72 hours,surgery or trauma within past 3 months, current malignancy), personal or family history of VTE (percentage of members with VTE history La Presse Médicale - 1493
A
R T I C L E
Chlamydia pneumoniae IgG serological status and venous thromboembolism
O R I G I N A L
A cross-sectional hospital based study
among first-degree relatives).We measured the fibrinogen level by the usual method and asked for the smoking status (package/year), because these are wellknown arterial risk factors which are potentially linked 9-12 with chronic CP infection .
deep vein thrombosis (DVT) subgroup (aggregation of distal and PVT groups) to the others (without VTE, with PE alone, with superficial and/or muscular vein thrombosis groups); lastly we studied the PVT positive patients versus all others. For continuous variables, the groups were compared using a Chi-square test or Anova,and for discreet variables using a Fischer’s exact test.When the comparison for CP status reached statistical significance (p≤0.05), an odds-ratio (OR) was calculated and adjusted, according to Wald's test, by logistic regression analysis to well-known risk factors of VTE and to classical factors of CP seropositivity: gender and age (<50 years, between 50 and 70 years, and >70 years).
DIAGNOSIS OF VTE All patients underwent objective tests within 48 hours of admission to confirm or rule out the diagnosis as pre13,14 .The standard procedures permitviously described ted classification of the patients as follows:without VTE, with at least proximal deep vein thrombosis (PVT),with at least distal deep vein thrombosis, with PE alone, and with superficial and/or muscular vein thrombosis.
Results
CP SEROLOGICAL STATUS DETERMINATION
MAIN CHARACTERISTICS OF THE POPULATION
Venous blood samples were collected within 24 hours following admission and a peptide Elisa (pep-Elisa),with Serochlamydia PEP Chlamydia pneumoniae IgG kit (Servibio, Meudon, France).The analysis was carried out blind at the end of the study by the same biologist, as indicated by the manufacturer. Each plasma was incubated in a well coated synthetic peptide specific to CP, then,after washing,with goat anti-human IgG.Lastly,after blocking, a positivity index (PI) calculated according to the absorbance at 405 nm determined the CP status (<0.9: negative, ≥0.9: positive).
From March 1992 to October 1999, 1193 patients were registered for suspected VTE.Among the 1010 samples available, 499 patients were VTE negative and 511 VTE positive (268 proximal,121 distal,27 muscular,29 superficial vein thrombosis and 66 PE without venous thrombosis).The main characteristics (table 1) of the VTE negative and the VTE positive groups were not statistically different except for age, well-known risk factor of VTE, and previous VTE.
CP SEROLOGICAL STATUS
STATISTICAL ANALYSIS
Seventy-nine patients were CP positive with a higher frequency of females (p=0.04) and smokers (p=0.02).We did not find any relationship between CP status and any other clinical risk factor for venous thrombosis.
We conducted an analysis of all patients (SPSS 10.0,SPSS Inc, Chicago, USA), according to VTE status (VTE negative patients versus all other patients).We compared the Table 1
Main characteristics of 1193 consecutive unselected outpatients referred to us for clinical suspicion of VTE and VTE status groups n Age, mean in years (SD)
Available
VTE + group
VYE - group
DVT group
PVT group
1010
511
499
389
268
Not available 183
63.3 (18.3)
65.1* (17.6)
61.3 (18.9)
65.4*(17.2)
66.7*(16.5)
63.3 (18.3)
Male
474 (46.9)
243 (47.6)
231 (46.3)
198* (50.8)
135 (50.3)
82 (44.8)
Previous VTE
253 (25.0)
172* (33.7)
81 (16.3)
131* (33.7)
96* (35.8)
45 (24.6)
Family history of VTE (SD) (n=355)
3.2 (8.3)
3.5 (8.5)
2.9 (8.4)
3.5 (7.8)
3.8 (7.8)
5.0 (10.3)
Current malignancy
106 (10.5)
73* (14.7)
33 (7.3)
57* (16.3)
48* (19.9)
19 (10.4)
Immobility >72h
238 (23.6)
143* (30.0)
95 (19.9)
111* (28.5)
75* (28.8)
44 (24.0)
Surgery or trauma within 3 months
102 (10.1)
63* (12.3)
39 (8.1)
53* (13.6)
26 (10.0)
15 (8.2)
Well-known risk factors
379 (37.5)
230* (45.0)
149 (29.8)
180* (46.3)
118* (44.0)
Chlamydia pneumoniae seropositivity
79 (7.8)
40 (7.8)
39 (7.8)
35 (9.0)
29* (10.8)
Smokers, mean in package/year (SD)
9.7 (17.7)
9.1 (16.9)
10.4 (18.5)
9.1 (17.6)
7.8* (16.1)
9.9 (16.9)
Fibrinogen, mean in g/L (n=876)
4.7 (3.6)
4.5 (1.8)
4.9 (4.7)
4.3* (1.8)
4.6 (1.6)
4.4* (1.8)
68 (37.2)
VTE: Venous Thrombosis and/or Embolism group, DVT: Deep Vein Thrombosis group, PVT: Proximal Deep Vein Thrombosis group. *p<0.05.
1494 - La Presse Médicale
4 décembre 2004 • tome 33 • n°21
L. de Saint Martin, E. Pasquier, F. Betsou, A.Tran, F. Couturaud, J. Orfila
ANALYSIS OF A LINK BETWEEN CP AND VTE STATUS We did not find any significant link between CP serological status and VTE or DVT status, but we identified a statistical difference regarding CP seropositivity between the PVT positive and negative groups (p=0.04, OR: 1.68 CI95% 1.04 to 2.72)
Discussion Our study failed to demonstrate any obvious link between CP serological status and VTE when considering unselected outpatients referred for clinical suspicion of VTE.The 7,8 discrepancy between the 3 studies (the 2 prior studies and ours [table 2]) may be explained by: • the heterogeneous serological methods used. The pep-Elisa test reached 70.7% concordance with the MIF 15 IgG assay , with a sensitivity and a specificity indicated by the manufacturer of 52.7% and 93.2% respectively. Its cross-reactions with other Chlamydia infections, specially Chlamydia trachomatis, have been demonstra16-19 . Howerer, the clinical relevance of CP serology ted results, independently of the test used, still remains unk20 nown ; • the choice of control group. In our study, the VTE control group is a “disease control”with a heterogeneous assortment of other conditions accounting for their symptoms. Comparison between VTE patients and heal7,8 thy controls , is more sensitive to detect an association, but does not avoid the confusing bias due to the presence of any underlying disease. In contrast, the choice
of the DVT and PVT controls could be more questionable, because they include some patients with venous thrombosis or PE. The main adverse effect of this approach could be the increase in CP seropositivity frequency in the control groups and therefore the reduced sensitivity of the study to detect any association; epidemiology. Contrasting with prior • the local 21,22 , females and smokers were more prevalent reports
W H AT
WAS KNOWN
Chlamydia pneumoniae serological status is associated with atherosclerosis • Among the several suggested mechanisms, a procoagulant effect is evoked • A link between Chlamydia pneumoniae serology and venous thromboembolism may be an argument for this hypothesis, despite two prior conflicting studies.
•
W H AT
THIS STUDY ADDS
• Any obvious link between Chlamydia pneumoniae serology and venous thromboembolism is demonstrated • The only association with proximal deep vein thrombosis observed is minor (OR<2) • The hypothesis of a procoagulant effect of chronic Chlamydia pneumoniae infection is irrelevant.
Table 2
Characteristics and results of Chlamydia pneumoniae seroepidemiological studies in VTE 7
8
Lozinguez
Koster
Patient eligibility
DVT or PE
DVT
PVT
DVT
VTE
Exclusion criteria
> 61 years
Cancer, > 70 years
no
No
no
routine check-up
Relatives
PVT excluded
DVT
Control group
Our study
excluded
VTE excluded
Matched
age and gender
age and gender
Suspicion of VTE
CP serology
MIF IgG >=256
ELISA IgG
pep ELISA IgG
delay/VTE*
a few months
19 months
<48 hours
Patients (% positif)
176 (54%)
474 (80%)
268(11%)
389 (9%)
511 (8%)
Controls (% positif)
197 (16%)
474 (78%)
742 (7%)
621 (7%)
499 (8%)
7.5
1.1
1.7
1.3
1.0
(4.4-12.5)
(0.9-1.4)
(1.0-2.8)
(0.8-2.1)
(0.62-1.6)
FV Leiden and FII G20210A mutations
fibrinogen, CRP, and clotting-factorVIII
OR adjusted CI95% according to
VTE risk factors: gender, and age
* time elapsed from VTE diagnosis to blood sample collection ; VTE: Venous Thrombosis and/or Embolism group 7
Lozinguez O, Arnaud E, Belec L et al. Demonstration of an association between Chlamydia pneumoniae infection and venous thromboembolic disease. Thromb Haemost 2000; 83: 887-91.
8
Koster T, Rosendaal F, Lieuw-an-Len D, Kroes A, Emmerich J, van Dissel J. Chlamydia pneumoniae IgG seropositivity and risk of deep-vein thrombosis. Lancet 2000; 355: 1694-5.
4 décembre 2004 • tome 33 • n°21
La Presse Médicale - 1495
A
R T I C L E
Chlamydia pneumoniae IgG serological status and venous thromboembolism
O R I G I N A L
A cross-sectional hospital based study
in the case group in our study. However, the percentage of CP seropositivity (8%), lower than expected (about 15%), decreased the strength of our study; • the VTE diagnosis procedure. This is unlikely because the sensitivity and specificity of the diagnostic tests performed were well-known and reliable, notably at the proximal site. Nevertheless, the observed association between proximal deep vein thrombosis and CP serological status suggests that the presence of CP antibodies might be a minor risk factor for venous thrombosis.The interpretation of an OR below two should be cautious but 35 of the 39 positive patients for CP and VTE belonged to the PVT positive group and the hypothesis of a selection bias
is unlikely because:
• the main characteristics of the two groups are almost identical (table 1);
• the OR remains significant, 1.70 (CI95% 1.05 to 2.77), even after stratification according to well-known risk factors of VTE: gender and age; • the design of the study would tend to underestimate a potential link. The question,as in the atherosclerosis–CP debate,should be:“causative agent or innocent bystander?”.Other studies should be carried out to specify the mechanisms, such as the increase of factor VIII level, which was suggested 23 in CMV infection ,or elevated plasma concentrations of 24 IL-6 and IL-8 . ■
References 1 Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? [see comments]. Lancet 1997; 350: 430-6. 2 Campbell LA, Rosenfeld M, Kuo CC. The role of chlamydia pneumoniae in atherosclerosis - recent evidence from animal models [In Process Citation]. Trends Microbiol 2000; 8: 255-57. 3 Fong IW. Value of animal models for Chlamydia pneumoniae-related atherosclerosis. Am Heart J 1999; 138: S512-S513. 4 Muhlestein JB. Chlamydia pneumoniae-Induced Atherosclerosis in a Rabbit Model. J Infect Dis 2000; 181: S505-S507. 5 Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research. Circulation 1997; 96: 4095-103. 6 Fryer RH, Schwobe EP, Woods ML, Rodgers GM. Chlamydia species infect human vascular endothelial cells and induce procoagulant activity. J Investig Med 1997; 45: 168-74. 7 Lozinguez O, Arnaud E, Belec L et al. Demonstration of an association between Chlamydia pneumoniae infection and venous thromboembolic disease. Thromb Haemost 2000; 83: 887-91. 8 Koster T, Rosendaal F, Lieuw-an-Len D, Kroes A, Emmerich J, van Dissel J. Chlamydia pneumoniae IgG seropositivity and risk of deep-vein thrombosis. Lancet 2000; 355: 1694-5. 9 Ernst E, Resch KL. Fibrinogen as a cardiovascular risk
1496 - La Presse Médicale
10
11
12
13
14 15
16
17
factor: a meta-analysis and review of the literature. Ann Intern Med 1993; 118: 956-63. Assman G, Cullen P, Shulte H. The Münster Heart study (PROCAM). Results of follow-up at 8 years. Eur Heart J 1998; 19: A2-A11. Hahn DL, Golubjatnikov R. Smoking is a potential confounder of the Chlamydia pneumoniae-coronary artery disease association. Arterioscler Thromb 1992; 12: 945-7. Toss H, Gnarpe J, Gnarpe H, Siegbahn A, Lindahl B, Wallentin L. Increased fibrinogen levels are associated with persistent Chlamydia pneumoniae infection in unstable coronary artery disease. Eur Heart J 1998; 19: 570-7. Leroyer C, Mercier B, Escoffre M. Factor V Leiden prevalence in venous thromboembolism patients. Chest 1997; 111: 1603-6. PIOPED investigators. Value of the ventilation/perfusion scan in acute pulmonary. JAMA 1990; 263: 2753-9. Maraha B, den Heijer M, Peeters M. Chlamydia pneumoniae IgG seropositivity in deep-vein thrombosis [letter]. Lancet 2000; 356: 1606-7. Gonen R, Shemer-Avni Y, Csango P, Sarov B, Friedman M. Serum activity to Chlamydia trachomatis and C. pneumoniae antigens in patients with documented infection and in healthy children by immunofluorescence and immunoblotting techniques. APMIS 1993; 101: 719-26. Kern DG, Neill MA, Schachter J. A seroepidemiologique study of Chlamydia pneumoniae in Rhode-Island -
18
19
20
21
22 23
24
evidence of serology cross-reactivity. Chest 1993; 104: 208-13. Moss TR, Darougar S, Woodland RM, Nathan M, Dines RJ, Cathine V. Antibodies to Chlamydia species in patients attending a genitourinary clinic and the impact of antibodies to Chlamydia pneumoniae and Chlamydia psittaci on the sensisitivity and the specificity of Chlamydia trachomatis serology tests. Sexuallity Transmitted Diseases 1993; 20: 61-5. Maurin M, Etienne J, Raoult D. Serological cross-reaction between Bartonella and Chlamydia species: implications for diagnosis. J Clin Microbiol 1997; 35: 2287. Tuuminen T., Palomaki P., Paavonen J. The use of serologic tests for the diagnosis of chlamidial infections. J Microbiol Methods 2000; 42: 265-79. Karvonen M, Tuomilehto J, Pitkaniemi J. Chlamydia pneumoniae IgG antibody prevalence in south western and eastern Finland in 1982 and 1987. Int J Epidemiol 1994; 26: 176-84. Kuo C, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 1995; 8: 451-61. Schambeck CM, Hinney K, Gleixner J, Keller F. Venous thromboembolism and associated high plasma factor VIII levels: linked to cytomegalovirus infection?[letter]. Thromb Haemost 2000; 83: 510-11. Maraha B, Peeters MF, van Aken BE, den Heijer M. Chlamydia pneumoniae, systemic inflammation and the risk of venous thrombosis. Diagn Microbiol Infect Dis 2002; 42: 153-7.
4 décembre 2004 • tome 33 • n°21
L. de Saint Martin1, 1 2 E. Pasquier , F. Betsou , 3 1 A.Tran , F. Couturaud , 2 J. Orfila
1 - Internal Medicine and Chest Disease Department, University Hospital of Brest, France 2 - Biobanque of Picardie, Amiens, France 3 - Department of Microbiology, University Hospital of Brest, France Oral communication: 40th Interscience Conference on Antimicrobial Agents and Chemotheray, Sept 2000, Toronto, Abstract: 1985.
Corresponding author: L. de Saint Martin, Hôpital de la Cavale
A
© 2004, Masson, Paris
R T I C L E
O R I G I N A L
Chlamydia pneumoniae IgG serological status and venous thromboembolism A cross-sectional hospital based study
Summary
Résumé
Objective To search for a link between Chlamydia pneumoniae serological status and venous thromboembolic disease. Methods From March 1992 to October 1999, we conducted a crosssectional hospital-based study of consecutive unselected outpatients referred to us for clinical suspicion of venous thromboembolism. We compared the Chlamydia pneumoniae serological status with respectively, the venous thromboembolism, the deep vein thrombosis and the proximal deep vein thrombosis status. Results Among 1193 patients registered for suspected venous thromboembolism, 1010 samples were available (499 negative and 511 positive patients for venous thromboembolism). Seventy-nine patients were Chlamydia pneumoniae positive. Our work failed to demonstrate any clear association between Chlamydia pneumoniae and venous thromboembolism status. Nevertheless, we identified a statistical difference regarding Chlamydia pneumoniae seropositivity and proximal vein thrombosis status (adjusted odds ratio of 1.70, CI95%: 1.05 to 2.77). Conclusion The presence of Chlamydia pneumoniae antibodies might be a minor risk factor for venous thrombosis.
Statut sérologique IgG à l’égard de Chlamydia pneumoniae et maladie veineuse thromboembolique
L. de Saint Martin, E. Pasquier, F. Betsou, A. Tran, F. Couturaud, J. Orfila Presse Med 2004; 33: 1493-6 © 2004, Masson, Paris
Blanche, 29609 Brest cedex, France Tel: 02 98 34 73 36 Fax: 02 98 34 79 44 [email protected]
Étude d’un échantillon de patients hospitalisés Objectif Chercher un lien entre statut sérologique à l’égard de Chlamydia pneumoniae et maladie veineuse thromboembolique. Méthodes De mars 1992 à octobre 1999 tous les patients consécutifs hospitalisés pour suspicion clinique de maladie veineuse thromboembolique ont été inclus. Leur statut sérologique à l’égard de Chlamydia pneumoniae a été comparé respectivement au statut veineux thromboembolique, à l’existence d’une thrombose veineuse profonde et à celle d’une thrombose veineuse proximale. Résultats 1193 patients ont été admis pour suspicion de maladie veineuse thromboembolique ; 1 010 échantillons ont été disponibles (499 patients n’ayant pas de maladies veineuse thromboembolique et 511 en étant atteints). 79 patients avaient une sérologie positive pour Chlamydia pneumoniae. Il n’a pas été montré d’association entre le statut pour C. pneumoniae et l’état thromboembolique veineux. Néanmoins, il a été trouvé une différence statistique concernant la séropositivité pour C. pneumoniae et l’existence d’une thrombose veineuse proximale (odds ratio ajusté 1,70, intervalle de confiance 95 % : 1,05 à 2,77). Conclusion La présence d’anticorps à l’égard de Chlamydia pneumoniae peut être un facteur de risque minime de thrombose veineuse.
Received February, 5, 2003 Accepted April, 8, 2004
A
large number of seroepidemiological studies1, 2-4 confirmed by other data , have suggested an association between chronic Chlamydia pneumoniae (CP) infection and atherosclerosis. Several mechanisms of CP action have been suggested: endothelial injury,antigenic mimicry,increase of standard risk factors of atherosclerosis (dyslipidemia, body mass index), and/or procoagulant effects (dysfunction of pla1,5,6 telets or coagulation proteins) . In the event of coagulation protein dysfunction, a link between CP and venous thromboembolism (VTE) may be conceivable. Because the two previous seroepide7,8 miological studies gave conflicting results , we explored CP status in VTE patients.
4 décembre 2004 • tome 33 • n°21
Methods PARTICIPANTS From March 1992 to October 1999, all consecutive patients with a clinically suspected VTE admitted to the Internal Medicine and Chest Disease Department (University Hospital of Brest, Brittany, EU) were included.
CLINICAL AND BIOLOGICAL DATA All patients underwent a complete clinical work-up concerning well-known risk factors for VTE (prolonged immobilisation >72 hours,surgery or trauma within past 3 months, current malignancy), personal or family history of VTE (percentage of members with VTE history La Presse Médicale - 1493
A
R T I C L E
Chlamydia pneumoniae IgG serological status and venous thromboembolism
O R I G I N A L
A cross-sectional hospital based study
among first-degree relatives).We measured the fibrinogen level by the usual method and asked for the smoking status (package/year), because these are wellknown arterial risk factors which are potentially linked 9-12 with chronic CP infection .
deep vein thrombosis (DVT) subgroup (aggregation of distal and PVT groups) to the others (without VTE, with PE alone, with superficial and/or muscular vein thrombosis groups); lastly we studied the PVT positive patients versus all others. For continuous variables, the groups were compared using a Chi-square test or Anova,and for discreet variables using a Fischer’s exact test.When the comparison for CP status reached statistical significance (p≤0.05), an odds-ratio (OR) was calculated and adjusted, according to Wald's test, by logistic regression analysis to well-known risk factors of VTE and to classical factors of CP seropositivity: gender and age (<50 years, between 50 and 70 years, and >70 years).
DIAGNOSIS OF VTE All patients underwent objective tests within 48 hours of admission to confirm or rule out the diagnosis as pre13,14 .The standard procedures permitviously described ted classification of the patients as follows:without VTE, with at least proximal deep vein thrombosis (PVT),with at least distal deep vein thrombosis, with PE alone, and with superficial and/or muscular vein thrombosis.
Results
CP SEROLOGICAL STATUS DETERMINATION
MAIN CHARACTERISTICS OF THE POPULATION
Venous blood samples were collected within 24 hours following admission and a peptide Elisa (pep-Elisa),with Serochlamydia PEP Chlamydia pneumoniae IgG kit (Servibio, Meudon, France).The analysis was carried out blind at the end of the study by the same biologist, as indicated by the manufacturer. Each plasma was incubated in a well coated synthetic peptide specific to CP, then,after washing,with goat anti-human IgG.Lastly,after blocking, a positivity index (PI) calculated according to the absorbance at 405 nm determined the CP status (<0.9: negative, ≥0.9: positive).
From March 1992 to October 1999, 1193 patients were registered for suspected VTE.Among the 1010 samples available, 499 patients were VTE negative and 511 VTE positive (268 proximal,121 distal,27 muscular,29 superficial vein thrombosis and 66 PE without venous thrombosis).The main characteristics (table 1) of the VTE negative and the VTE positive groups were not statistically different except for age, well-known risk factor of VTE, and previous VTE.
CP SEROLOGICAL STATUS
STATISTICAL ANALYSIS
Seventy-nine patients were CP positive with a higher frequency of females (p=0.04) and smokers (p=0.02).We did not find any relationship between CP status and any other clinical risk factor for venous thrombosis.
We conducted an analysis of all patients (SPSS 10.0,SPSS Inc, Chicago, USA), according to VTE status (VTE negative patients versus all other patients).We compared the Table 1
Main characteristics of 1193 consecutive unselected outpatients referred to us for clinical suspicion of VTE and VTE status groups n Age, mean in years (SD)
Available
VTE + group
VYE - group
DVT group
PVT group
1010
511
499
389
268
Not available 183
63.3 (18.3)
65.1* (17.6)
61.3 (18.9)
65.4*(17.2)
66.7*(16.5)
63.3 (18.3)
Male
474 (46.9)
243 (47.6)
231 (46.3)
198* (50.8)
135 (50.3)
82 (44.8)
Previous VTE
253 (25.0)
172* (33.7)
81 (16.3)
131* (33.7)
96* (35.8)
45 (24.6)
Family history of VTE (SD) (n=355)
3.2 (8.3)
3.5 (8.5)
2.9 (8.4)
3.5 (7.8)
3.8 (7.8)
5.0 (10.3)
Current malignancy
106 (10.5)
73* (14.7)
33 (7.3)
57* (16.3)
48* (19.9)
19 (10.4)
Immobility >72h
238 (23.6)
143* (30.0)
95 (19.9)
111* (28.5)
75* (28.8)
44 (24.0)
Surgery or trauma within 3 months
102 (10.1)
63* (12.3)
39 (8.1)
53* (13.6)
26 (10.0)
15 (8.2)
Well-known risk factors
379 (37.5)
230* (45.0)
149 (29.8)
180* (46.3)
118* (44.0)
Chlamydia pneumoniae seropositivity
79 (7.8)
40 (7.8)
39 (7.8)
35 (9.0)
29* (10.8)
Smokers, mean in package/year (SD)
9.7 (17.7)
9.1 (16.9)
10.4 (18.5)
9.1 (17.6)
7.8* (16.1)
9.9 (16.9)
Fibrinogen, mean in g/L (n=876)
4.7 (3.6)
4.5 (1.8)
4.9 (4.7)
4.3* (1.8)
4.6 (1.6)
4.4* (1.8)
68 (37.2)
VTE: Venous Thrombosis and/or Embolism group, DVT: Deep Vein Thrombosis group, PVT: Proximal Deep Vein Thrombosis group. *p<0.05.
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L. de Saint Martin, E. Pasquier, F. Betsou, A.Tran, F. Couturaud, J. Orfila
ANALYSIS OF A LINK BETWEEN CP AND VTE STATUS We did not find any significant link between CP serological status and VTE or DVT status, but we identified a statistical difference regarding CP seropositivity between the PVT positive and negative groups (p=0.04, OR: 1.68 CI95% 1.04 to 2.72)
Discussion Our study failed to demonstrate any obvious link between CP serological status and VTE when considering unselected outpatients referred for clinical suspicion of VTE.The 7,8 discrepancy between the 3 studies (the 2 prior studies and ours [table 2]) may be explained by: • the heterogeneous serological methods used. The pep-Elisa test reached 70.7% concordance with the MIF 15 IgG assay , with a sensitivity and a specificity indicated by the manufacturer of 52.7% and 93.2% respectively. Its cross-reactions with other Chlamydia infections, specially Chlamydia trachomatis, have been demonstra16-19 . Howerer, the clinical relevance of CP serology ted results, independently of the test used, still remains unk20 nown ; • the choice of control group. In our study, the VTE control group is a “disease control”with a heterogeneous assortment of other conditions accounting for their symptoms. Comparison between VTE patients and heal7,8 thy controls , is more sensitive to detect an association, but does not avoid the confusing bias due to the presence of any underlying disease. In contrast, the choice
of the DVT and PVT controls could be more questionable, because they include some patients with venous thrombosis or PE. The main adverse effect of this approach could be the increase in CP seropositivity frequency in the control groups and therefore the reduced sensitivity of the study to detect any association; epidemiology. Contrasting with prior • the local 21,22 , females and smokers were more prevalent reports
W H AT
WAS KNOWN
Chlamydia pneumoniae serological status is associated with atherosclerosis • Among the several suggested mechanisms, a procoagulant effect is evoked • A link between Chlamydia pneumoniae serology and venous thromboembolism may be an argument for this hypothesis, despite two prior conflicting studies.
•
W H AT
THIS STUDY ADDS
• Any obvious link between Chlamydia pneumoniae serology and venous thromboembolism is demonstrated • The only association with proximal deep vein thrombosis observed is minor (OR<2) • The hypothesis of a procoagulant effect of chronic Chlamydia pneumoniae infection is irrelevant.
Table 2
Characteristics and results of Chlamydia pneumoniae seroepidemiological studies in VTE 7
8
Lozinguez
Koster
Patient eligibility
DVT or PE
DVT
PVT
DVT
VTE
Exclusion criteria
> 61 years
Cancer, > 70 years
no
No
no
routine check-up
Relatives
PVT excluded
DVT
Control group
Our study
excluded
VTE excluded
Matched
age and gender
age and gender
Suspicion of VTE
CP serology
MIF IgG >=256
ELISA IgG
pep ELISA IgG
delay/VTE*
a few months
19 months
<48 hours
Patients (% positif)
176 (54%)
474 (80%)
268(11%)
389 (9%)
511 (8%)
Controls (% positif)
197 (16%)
474 (78%)
742 (7%)
621 (7%)
499 (8%)
7.5
1.1
1.7
1.3
1.0
(4.4-12.5)
(0.9-1.4)
(1.0-2.8)
(0.8-2.1)
(0.62-1.6)
FV Leiden and FII G20210A mutations
fibrinogen, CRP, and clotting-factorVIII
OR adjusted CI95% according to
VTE risk factors: gender, and age
* time elapsed from VTE diagnosis to blood sample collection ; VTE: Venous Thrombosis and/or Embolism group 7
Lozinguez O, Arnaud E, Belec L et al. Demonstration of an association between Chlamydia pneumoniae infection and venous thromboembolic disease. Thromb Haemost 2000; 83: 887-91.
8
Koster T, Rosendaal F, Lieuw-an-Len D, Kroes A, Emmerich J, van Dissel J. Chlamydia pneumoniae IgG seropositivity and risk of deep-vein thrombosis. Lancet 2000; 355: 1694-5.
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A
R T I C L E
Chlamydia pneumoniae IgG serological status and venous thromboembolism
O R I G I N A L
A cross-sectional hospital based study
in the case group in our study. However, the percentage of CP seropositivity (8%), lower than expected (about 15%), decreased the strength of our study; • the VTE diagnosis procedure. This is unlikely because the sensitivity and specificity of the diagnostic tests performed were well-known and reliable, notably at the proximal site. Nevertheless, the observed association between proximal deep vein thrombosis and CP serological status suggests that the presence of CP antibodies might be a minor risk factor for venous thrombosis.The interpretation of an OR below two should be cautious but 35 of the 39 positive patients for CP and VTE belonged to the PVT positive group and the hypothesis of a selection bias
is unlikely because:
• the main characteristics of the two groups are almost identical (table 1);
• the OR remains significant, 1.70 (CI95% 1.05 to 2.77), even after stratification according to well-known risk factors of VTE: gender and age; • the design of the study would tend to underestimate a potential link. The question,as in the atherosclerosis–CP debate,should be:“causative agent or innocent bystander?”.Other studies should be carried out to specify the mechanisms, such as the increase of factor VIII level, which was suggested 23 in CMV infection ,or elevated plasma concentrations of 24 IL-6 and IL-8 . ■
References 1 Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? [see comments]. Lancet 1997; 350: 430-6. 2 Campbell LA, Rosenfeld M, Kuo CC. The role of chlamydia pneumoniae in atherosclerosis - recent evidence from animal models [In Process Citation]. Trends Microbiol 2000; 8: 255-57. 3 Fong IW. Value of animal models for Chlamydia pneumoniae-related atherosclerosis. Am Heart J 1999; 138: S512-S513. 4 Muhlestein JB. Chlamydia pneumoniae-Induced Atherosclerosis in a Rabbit Model. J Infect Dis 2000; 181: S505-S507. 5 Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: an assessment of the evidence and need for future research. Circulation 1997; 96: 4095-103. 6 Fryer RH, Schwobe EP, Woods ML, Rodgers GM. Chlamydia species infect human vascular endothelial cells and induce procoagulant activity. J Investig Med 1997; 45: 168-74. 7 Lozinguez O, Arnaud E, Belec L et al. Demonstration of an association between Chlamydia pneumoniae infection and venous thromboembolic disease. Thromb Haemost 2000; 83: 887-91. 8 Koster T, Rosendaal F, Lieuw-an-Len D, Kroes A, Emmerich J, van Dissel J. Chlamydia pneumoniae IgG seropositivity and risk of deep-vein thrombosis. Lancet 2000; 355: 1694-5. 9 Ernst E, Resch KL. Fibrinogen as a cardiovascular risk
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10
11
12
13
14 15
16
17
factor: a meta-analysis and review of the literature. Ann Intern Med 1993; 118: 956-63. Assman G, Cullen P, Shulte H. The Münster Heart study (PROCAM). Results of follow-up at 8 years. Eur Heart J 1998; 19: A2-A11. Hahn DL, Golubjatnikov R. Smoking is a potential confounder of the Chlamydia pneumoniae-coronary artery disease association. Arterioscler Thromb 1992; 12: 945-7. Toss H, Gnarpe J, Gnarpe H, Siegbahn A, Lindahl B, Wallentin L. Increased fibrinogen levels are associated with persistent Chlamydia pneumoniae infection in unstable coronary artery disease. Eur Heart J 1998; 19: 570-7. Leroyer C, Mercier B, Escoffre M. Factor V Leiden prevalence in venous thromboembolism patients. Chest 1997; 111: 1603-6. PIOPED investigators. Value of the ventilation/perfusion scan in acute pulmonary. JAMA 1990; 263: 2753-9. Maraha B, den Heijer M, Peeters M. Chlamydia pneumoniae IgG seropositivity in deep-vein thrombosis [letter]. Lancet 2000; 356: 1606-7. Gonen R, Shemer-Avni Y, Csango P, Sarov B, Friedman M. Serum activity to Chlamydia trachomatis and C. pneumoniae antigens in patients with documented infection and in healthy children by immunofluorescence and immunoblotting techniques. APMIS 1993; 101: 719-26. Kern DG, Neill MA, Schachter J. A seroepidemiologique study of Chlamydia pneumoniae in Rhode-Island -
18
19
20
21
22 23
24
evidence of serology cross-reactivity. Chest 1993; 104: 208-13. Moss TR, Darougar S, Woodland RM, Nathan M, Dines RJ, Cathine V. Antibodies to Chlamydia species in patients attending a genitourinary clinic and the impact of antibodies to Chlamydia pneumoniae and Chlamydia psittaci on the sensisitivity and the specificity of Chlamydia trachomatis serology tests. Sexuallity Transmitted Diseases 1993; 20: 61-5. Maurin M, Etienne J, Raoult D. Serological cross-reaction between Bartonella and Chlamydia species: implications for diagnosis. J Clin Microbiol 1997; 35: 2287. Tuuminen T., Palomaki P., Paavonen J. The use of serologic tests for the diagnosis of chlamidial infections. J Microbiol Methods 2000; 42: 265-79. Karvonen M, Tuomilehto J, Pitkaniemi J. Chlamydia pneumoniae IgG antibody prevalence in south western and eastern Finland in 1982 and 1987. Int J Epidemiol 1994; 26: 176-84. Kuo C, Jackson LA, Campbell LA, Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 1995; 8: 451-61. Schambeck CM, Hinney K, Gleixner J, Keller F. Venous thromboembolism and associated high plasma factor VIII levels: linked to cytomegalovirus infection?[letter]. Thromb Haemost 2000; 83: 510-11. Maraha B, Peeters MF, van Aken BE, den Heijer M. Chlamydia pneumoniae, systemic inflammation and the risk of venous thrombosis. Diagn Microbiol Infect Dis 2002; 42: 153-7.
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