Transfusion Medicine, 2010, 20, 237–243

doi: 10.1111/j.1365-3148.2010.01003.x

ORIGINAL ARTICLE

Detection of Chlamydia pneumoniae in peripheral blood mononuclear cells of healthy blood donors in Tehran Regional Educational Blood Transfusion Centre Gh. Karimi,1 Sh. Samiei,1 H. Hatami,2 A. Gharehbaghian,1 V. Vafaiyan1 & M. Tabrizi Namini1 1 Research

Centre of Iranian Blood Transfusion Organization, and 2 School of Public Health, Shahid Beheshti University of Medical Sciences,

Tehran, Iran Received 20 June 2009; accepted for publication 03 March 2010

summary. Chlamydia pneumoniae is a common pathogen in the world often causing upper or lower respiratory tract infection and may also be linked to some chronic inflammatory diseases. Recent studies have shown that a high percentage of healthy blood donors harbour Chlamydia DNA and antigens. The objective of this study was to investigate the presence of this microorganism among blood donors. Blood samples were collected between November 2004 and March 2005 from 196 healthy blood donors. Ten millilitre of blood was collected in ethylenediaminetetraacetic (EDTA) tube. Reverse transcription of RNA was performed with Moloney murine leukaemia virus (MMLV) reverse transcriptase and random primers hexamer. Polymerase chain reaction products were evaluated by electrophoresis. Data were analysed using the χ2 test and t-test. Of the 196 healthy blood donors, 7·1% were C. pneumoniae DNA positive (CI 95% = 3·51–10·69),

which is slightly higher in female (8·5%) than male (6·5%) donors; this difference was not found to be significant (P = 0·4). The average age of study groups was 40·84 (SD ± 10·80) years; significant association was not found between age groups and the presence of C. pneumoniae DNA. There was no significant differences between positive rate and first-time [37 (19·3%)] and repeat [155 (80·7%)] donors. C. pneumoniae DNA seems to be frequent in apparently healthy blood donors; therefore, it can be a threat for blood safety. But further studies are needed to evaluate the survival of C. pneumoniae in blood bank conditions and in blood recipients to define the clinical importance of such findings. Elimination of intracellular bacteria by filtration is an effective strategy for risk reduction.

Chlamydia are obligate intracellular bacteria including the four currently recognised species of C. psittaci, C. trachomatis, C. pneumoniae (Chlamydophila pneumoniae) and C. pecorum, which have been classified according to their antigenic, pathological and molecular properties (Jackson et al., 2005). Following reclassification, the order Chlamydiale currently comprises four families, namely, the Chlamydiaceae, Parachlamydiaceae, Simkaniaceae and Waddiaceae, that are divided into two genera, Chlamydia and Chlamydophila, and nine species (Everett et al., 1999; Bush & Everret, 2001). However, this reclassification is controversial in

C. pneumoniae community. C. pneumoniae, initially isolated from conjunctival specimens during trachoma studies in Taiwan in 1965 and Iran in 1968, is an extremely common pathogen in the world and often causes recurrent infection throughout life (Dwyer et al., 1972; Kuo et al., 1995; Schaehter et al., 2004). Seroepidemiological studies have shown that more than 50% (Brunham et al., 2008) of adults worldwide are infected with C. pnemoniae during their life time and most of these infections involve the upper or lower respiratory tract. Like other respiratory transmitted pathogens, C. pnemoniae is presumed to spread from person to person through respiratory droplets (Forbes et al., 2002); however, contact with contaminated surfaces may be a potential mode of transmission (Verkooyen et al., 1995). Moreover, other variables such as temperature and

Correspondence: A. Gharehbaghian, Research Centre of Iranian Blood Transfusion Organization, Hemmat Exp. Way, Tehran, Iran. Tel.: +98 21 88601599; fax: +98 21 88601599; e-mail: [email protected] © 2010 The Authors Journal compilation © 2010 British Blood Transfusion Society

Key words: blood donor, Chlamydia pneumoniae, peripheral blood mononuclear cells, polymerase chain reaction.

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238 Gh. Karimi et al. humidity have influence on survival of this bacterium in aerosols (Theunissen et al., 1993). Community-acquired pneumonia (CAP) to which C. pneumoniae contributes in 6% to more than 20% of cases still remains one of the most important causes of mortality and morbidity (Almirall et al., 1993; Miyashita et al., 2001, 2002; Koh et al., 2002; Monno et al., 2002; Song et al., 2008). Furthermore, recent evidence suggests that the organism may infect macrophages within the respiratory tract and use the blood stream for systemic dissemination (Airenne et al., 1999; Gieffers et al., 2004). It has been increasingly recognised that C. pneumoniae may be linked to some chronic inflammatory diseases, including sclerotic cardiovascular disease (Grayston et al., 2000; Bahrmand et al., 2004; Halfon et al., 2006; Kaplan et al., 2006; Romano et al., 2006; Khalili et al., 2007; Wang et al., 2007). An association with reactive arthritis (Inman et al., 2000; Villareal et al., 2002) and lung cancer (Littman et al., 2005) has been reported. There are some claims that C. pneumoniae is associated with acute and chronic neurological problems such as cerebrovascular accidents (Madre et al., 2002; Bucurescu et al., 2003), multiple sclerosis (Sriram et al., 1999; Stratton et al., 2006) and Alzheimer’s disease (Balin et al., 1998; Gerard et al., 2006); it has also been implicated in the pathobiology of asthma (Hahn et al., 2000; Ahmadi Torshizi et al., 2008). The potential of C. pneumoniae to act as a respiratory pathogen in immunocompromised patients has also been recognised (Gaydos et al., 1993). According to some studies (Kaukoranta-Tolvanen et al., 1996; Haranaga et al., 2001a), C. pneumoniae infection of lymphocytes and the ability to induce a cytokine response due to B and T lymphocyte cells and human peripheral blood mononuclear cells (PBMNCs) play an important role in the pathogenesis of the bacteria. Studies have shown that a high percentage of healthy blood donors harbour C. pneumoniae DNA and antigens within their PBMNCs (Boman et al., 1998; Rassu et al., 2001; Haranaga et al., 2001b). Nevertheless, some studies indicate a possible presence of viable C. pneumoniae in blood of healthy donors (Yamaguchi et al., 2004). All these studies show that C. pneumoniae may transmit through blood transfusion and endanger blood safety. Leucoreduction procedure by filtration is proposed as an effective method to reduce resident C. pneumoniae levels in RBC components (Ikejima et al., 2005). Studies investigating the presence of C. pneumoniae in blood donors have not been carried out so far in our country. This study was thus aimed to detect this microorganism among healthy blood donors in Tehran Blood Transfusion Centre.

MATERIALS AND METHODS Blood specimens Blood samples were collected from 196 voluntary healthy blood donors (138 male and 58 female donors; median age 40 years; age range, 18–64 years) between November 2004 and March 2005 in Tehran Blood Transfusion Centre. Simple random sampling method was employed for the selection. Medical check-up and physical examination by a physician are mandatory to ascertain the health of blood donors. There were no signs and symptoms of upper or lower respiratory tract infection in our donors. Any evidence of respiratory tract infection and use of antibiotics during the final two days prior to the donation time are routine donor deferral criteria in our centre. In addition, we excluded donors who used antibiotics within 2 weeks before the study. All subjects agreed to participate in this study and signed the informed consent document. The study was approved by the Ethics Committee of Iranian Blood Transfusion Organization Research Centre. RNA preparation and RT-PCR The presence of C. pneumoniae in PBMNCs of healthy blood donors was assessed by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from the treated PBMNCs (RNA extraction kit, Tripure, Roche, Germany). The cDNA was prepared as directed by the manufacturer’s guide (Promega, Madison, WI, USA) with random hexamer and 200 U of Moloney murine leukaemia virus RNase H (MMLVH, Promega) in a final volume of 10 μL. The quality and quantity control of extracted RNA were assessed through gel electrophoresis and spectrophotometric analysis. RNA was treated with DNase and run in parallel with cDNA in RT-PCR. In addition, a housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified to control the inhibitory effect of RNA solution. We used Yamaguchi et al. (2004) primers for 16S rRNA. PCR was carried out in a master mix containing 10 μL cDNA, 1 μm primer, 0.2 mm dNTP, 2.5 mm Mg2+ , 10% Betaein (5 m) and 1.5 U Fast Start Taq polymerase (Roche). The thermal cycling conditions were 95 ◦ C for 5 min followed by 40 cycles at 95 ◦ C for 15 s, 60 ◦ C for 60 s and 72 ◦ C for 20 s. The PCR products were evaluated by electrophoresis on 2% agarose gels (Fig. 1). Statistical analysis The statistical analysis was performed using SPSS software (version 16). The presence of Chlamydia pneumoniae DNA with 95% confidence interval was

© 2010 The Authors Journal compilation © 2010 British Blood Transfusion Society, Transfusion Medicine, 20, 237–243

Chlamydia pneumoniae in PBMNCs of blood donors

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C. pnenumoniae DNA positivity was slightly higher in females [5 (8·5%)] than in males [9 (6·5%)]; this difference was not found to be significant (χ2 , P = 0·401). The association of C. pneumoniae detection with donation frequency and job category was analysed; there was no significant difference between first-time [37 (19·3%)] and repeat [155 (80·7%)] donors as far as C. pneumoniae DNA positivity was concerned (χ2 , P = 0·478, Table 2). Blood donors were divided into three job categories of housekeepers (35·5%), workers (21·4%) and civil agents (35·7%), with no significant difference (χ2 , P = 0·444).

500 bp 250 bp

DISCUSSION (a)

(b)

(c)

(d)

(e)

Fig. 1. Detection of C. pneumoniae DNA in healthy blood donors. Agarose gel electrophoresis. (a) Positive control. (b) Size marker (50 bp). (c) Positive sample (430 bp). (d) Negative sample. (e) Negative control (ddw).

calculated for age groups, different sexes, donation frequency and job categories. The χ2 tests and t-tests were performed and statistical significance was established at P value <0·05. RESULTS We evaluated the presence of C. pneumoniae DNA in 196 voluntary healthy blood donors, including 58 (30%) female and 138 (70%) male donors. Overall positivity was 7·1% (CI 95% = 3·51–10·69). The correlation of C. pneumoniae detection in blood donors with age and sex was analysed. The frequency of positive results (20%) was higher in the 18–24 years age group. No significant difference was observed between age groups [χ2 , P = 0·3, mean age of positive donors (n = 14) 39·93 ± 14·25 vs mean age of negative donors (n = 182) 40·48 ± 10·62; t-test, P = 0·856] and the presence of C. pneumoniae (Table 1). Table 1. Distribution of Chlamydia pneumoniae DNA in different age classes Age (years) 18–24 25–34 35–44 45–54 55–64 Total

n1 15 44 62 54 21 196

PCR+ (%) 3 2 3 4 2 14

(20) (4·5) (4·8) (7·4) (9·52) (7·14)

CI 95% −0·24–22·24 −1·625–10·625 −0·52–10·12 0·5–14·3 −3·03–22·07 3·51–10·69

There was no significant difference between positive rate and age groups, χ2 , P = 0.3. 1 n = Number of subjects.

Several agents including bacteria and viruses affect blood safety. Blood safety strategies have focused on preventing the transmission of previously known agents and specific tests have been in place for detecting the presence of hepatitis B virus, hepatitis C virus, HIV, human T-cell lymphotropic virus and Treponema pallidum, thereby reducing the risks of transmission of these agents by blood transfusion to very low levels (Pillonel et al., 2002; Zou et al., 2004; Amini KafiAbad et al., 2009). On the contrary, the risk of new emerging pathogens has increased because many of these pathogens are the viruses present in the general population. These pathogens cause acute and transient viraemia in asymptomatic potential blood donors whose infection is not detectable, thereby increasing the risk of transfusion-transmitted agents (Dunstan et al., 2008; Stramer et al., 2009). Thus, conducting studies to evaluate the effects of these agents on blood safety is desirable. One of these pathogens potentially transmissible by blood transfusion is Chlamydia pneumoniae (Leiby, 2003). Although C. pneumoniae is not a new pathogen, recent studies indicate that it can be a threat to blood safety; therefore, designing and conducting more research related to this bacterium in different communities is an effective solution for the enhancement of blood safety. In this study, we detected the presence of C. pneumoniae in PBMNCs of healthy blood donors using RT-PCR. This method is a highly sensitive technique described by other authors like Yamaguchi et al. (2004) and Haranaga et al. (2001a). In this study, C. pneumoniae was detected in 14 of 196 (7·1%) healthy blood donors. The prevalence of C. pneumoniae in healthy community of Iran has not yet been extensively studied and this is the first study in the blood donor population in Iran. However, much of the current information on the epidemiology of C. pneumoniae infection is derived from serological studies using the Microimmunofluorescence

© 2010 The Authors Journal compilation © 2010 British Blood Transfusion Society, Transfusion Medicine, 20, 237–243

240 Gh. Karimi et al. Table 2. Prevalence of C. pneumoniae DNA in PBMNCs obtained from healthy blood donors C. pneumoniae DNA-positive

Sex Male Female Donation type First time Repeated

Number positive/tested (% positive)

CI 95%

Mean age of positive donors (years)

Median age of positive donors (years)

9/138 (6·5%) 5/58 (8·6%)

2·4–10·6 1·4–15·8

43·67 ± 12·46 33·20 ± 16·17

44 25

4/37 (10·8%) 10/155 (6·5%)

0·8–20·8 2·62–10·38

33·25 ± 15·39 42·60 ± 13·66

34 42

There was no significant difference between positive rate and sex or donation type (χ2 , P = 0·401 and P = 0·478, respectively).

(MIF) assay. These studies indicate that exposure to C. pneumoniae is common and occurs throughout the world, with seroprevalence rates of more than 50% among adults in many countries. This rate continues to increase gradually with age, reaching approximately 75% in elderly persons. Although the detection of C. pneumoniae seropositivity does not prove the presence of viable C. pneumoniae, it is important to note that this extensive dispersal may be a threat to blood safety. Several recently published studies suggest that C. pneumoniae may represent a risk factor for blood safety; for example, Yamaguchi et al. (2004) investigated the prevalence of viable C. pneumoniae in PBMNCs of healthy blood donors [13 positive of 70 donors (18·5%)]. Kaul et al. (2000) found C. pneumoniae in 5 (26%) of 19 healthy blood donors using Nested PCR. Haranaga et al. (2001b) showed 8.9% positivity in PBMNCs of 237 blood samples by using PCR that was specific for C. pneumoniae 16S rRNA gene and by staining with FITC conjugated Chlamydiae MoAb. This result is nearly similar to that of our study. There is a very high prevalence in studies carried out by Rassu et al. (2001) and Boman et al. (1998), showing 46·15 and 46%, respectively. The difference in the prevalence of C. pneumoniae in PBMNCs reported by these studies may be due to differences in population and geographical distribution, or methodological differences. No significant difference was found concerning positive test results and age groups (P = 0 · 856). In this regard, our results are similar to some other studies (Rassu et al., 2001; Haranaga et al., 2001b; Yamaguchi et al., 2004). The younger donor age seems to have a positive, although insignificant, correlation with the prevalence of C. pneumoniae, as shown in our study and those of others (Rassu et al., 2001; Yamaguchi et al., 2004). In our study, C. pneumoniae positivity was slightly higher in females (8·6%) than in males (6·5%); there is no significant correlation between the

presence of C. pneumoniae and gender. Other studies also support the results (Rassu et al., 2001; Yamaguchi et al., 2004). We also evaluated the correlation between donation frequency and C. pneumoniae positivity. Our donors were categorised into two groups (first-time and repeat donors). Repeat donors have consistently been shown to have a lower incidence of infectious diseases and lower level risks than first-time donors (Williams et al., 1997; Glynn et al., 2000; Barreto et al., 2005; Kasraian & Troab Jahromi, 2007; Gharebaghian et al., 2008). In the current study, however, no correlation was found between donation frequency and C. pneumoniae existence (P = 0·478); therefore, the incidence of infection could be attributed to the fact that C. pneumoniae transmits through respiratory routes to which both groups in the community are equally exposed. According to existing evidence, the presence of C. pneumoniae in blood of apparently healthy donors has been demonstrated. But there is a lack of sufficient evidence on the probability of transmission to blood recipients and the pathogenesis of C. pneumoniae via blood transfusion. Thus, there is no recommendation for routine laboratory screening and blood donor exclusion criteria in donors without any clinical manifestation. As C. pneumoniae is an obligate intracellular pathogen, as mentioned previously, leucoreduction by filtration may be a practical action for the reduction of this pathogen. Ikejima et al. (2005) showed that leucoreduction by filtration is an effective method to reduce resident C. pneumoniae levels in RBC components significantly. Leucoreduction is a currently practiced policy in Iranian Blood Transfusion Organization to reduce transfusion-transmitted reactions in recipients. At present, prestorage leucoreduction of whole blood is the prevailing method in case of thalassaemics in all blood centres. Moreover, bedside filtration is used for highrisk patients, including neonates and immune-deficient patients.

© 2010 The Authors Journal compilation © 2010 British Blood Transfusion Society, Transfusion Medicine, 20, 237–243

Chlamydia pneumoniae in PBMNCs of blood donors In conclusion, according to this and other studies, the presence of C. pneumoniae DNA seems to be frequent in apparently healthy blood donors; therefore, it can be a threat to blood safety. Considerable improvements in blood safety have been made that can be attributed to national standards set for donor eligibility and exclusion, and to laboratory screening procedures being followed in Iran. Nevertheless, as for probable and unknown risks related to intracellular microorganisms such as C. pneumoniae, more use of leucoreduced products is recommended, especially for high-risk recipients. However, further studies are needed to evaluate the survival of C. pneumoniae in blood bank conditions; further meticulous studies on blood recipients are also required to define the clinical importance of such findings. ACKNOWLEDGEMENTS This study was supported by Iranian Blood Transfusion Organization Research Centre. We would like to thank our colleagues in Tehran Regional Educational Blood Transfusion Centre and the blood donors who participated in our research. DECLARATIONS OF INTEREST The authors declare that they have no conflict of interests. REFERENCES Ahmadi Torshizi, A., Tohidi, M., Attaran, D., Khaje Karamadin, M. & Ghazvini, K. (2008) Role of Chlamydia pneumoniae infection in asthma in northeast of Iran. Iranian Journal of Allergy, Asthma and Immunology, 7, 45–46. Airenne, S., Surcel, H.M., Alak¨arpp¨a, H., Laitinen, K., Paavonen, J., Saikku, P. & Laurila, A. (1999) Chlamydia pneumoniae infection in human monocytes. Infection and Immunity, 67, 1445–1449. Almirall, J., Morat´o, I., Riera, F. et al. (1993) Incidence of community acquired pneumonia and Chlamydia pneumoniae: a prospective multicentre study. European Respiratory Journal, 6, 14–18. Bahrmand, A.R., Bahadori, M., Hossaini, A., Velayati, A.A., Aghabozorgy, S., Shakoor, A. & Bakayev, V.V. (2004) Chlamydia pneumoniae DNA is more frequent in advanced than in mild atherosclerosis lesions. Scandinavian Journal of Infectious Diseases, 36, 119–123. Balin, B.J., Gerard, H.C., Arking, E.J. et al. (1998) Identification and localization of Chlamydia pneumoniae in the Alzheimer’s brain. Medical Microbiology and Immunology, 187, 23–42. Barreto, C.C., Sabino, E.C., Goncalez, T.T. et al. (2005) Prevalence, incidence, and residual risk of human immunodeficiency virus among community and replacement first-time blood donors in Sao Paulo, Brazil. Transfusion, 45, 1709–1714.

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