Depletion of Resident Chlamydia pneumoniae through Leukoreduction by Filtration of Blood for Transfusion
http://www.100md.com
微生物临床杂志 2005年第9期
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida 33612
Florida Blood Services, St. Petersburg, Florida 33742
Department of Basic Laboratory Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
ABSTRACT
Current studies indicate that a significant percentage of healthy blood donors carry Chlamydia pneumoniae in their blood. Although the clinical significance of such findings is unknown, eradication of such bacteria from blood components may contribute to transfusion safety. Deletion of C. pneumoniae in Red Blood Cell (RBC) units was accomplished through leukoreduction by filtration. The presence of bacteria in RBC units before and after leukoreduction was assessed by real-time PCR using primers specific for C. pneumoniae 16S rRNA. The eluates of filters used for leukoreduction were also assessed by PCR and immunostaining with fluorescein isothiocyanate-conjugated chlamydial monoclonal antibodies specific for C. pneumoniae determination. Nineteen of 30 RBC units tested showed the presence of C. pneumoniae DNA. Leukofiltration resulted in a marked reduction of leukocytes as well as C. pneumoniae in terms of bacterial number and positive rate for the bacteria. The eluates of filters showed trapped bacteria determined by both PCR and immunostaining assays. Thus, leukoreduction with a filter is an effective method to significantly reduce resident C. pneumoniae levels in RBC components but may not be completely sufficient for total eradication of this pathogen.
INTRODUCTION
Chlamydia (Chlamydophila) pneumoniae, an obligate intracellular bacterium, causes a variety of respiratory diseases (12). A number of studies indicate that this pathogen is associated not only with respiratory diseases but also with chronic inflammatory diseases, such as atherosclerosis, endocarditis, asthma, and arthritis (9, 13, 20, 23). It has been shown seroepidemiologically that 50 to 80% of the adult population has had prior exposure to this pathogen (5, 11, 27). Although the pathogenic potential of this microorganism in the respiratory system is well established, the pathogen may disseminate systemically from respiratory sites, probably via circulating leukocytes (15, 19). In fact, the presence of C. pneumoniae DNA in the peripheral blood of individuals with cardiovascular diseases as well as healthy subjects has been demonstrated in a significant percentage of these populations (3, 4, 6, 16-18, 22, 24, 25). In addition, another study by us revealed that the bacteria demonstrated in peripheral blood mononuclear cells obtained from healthy blood donors are viable with a readily detectable growth potential (29). Thus, the ubiquitous nature of chronic, persistent infection with C. pneumoniae and the presence of this pathogen in the blood of a potentially significant segment of the blood donor population may be an important consideration for transfusion medicine specialists.
The risk of complications associated with blood transfusion is a major concern to the general public. Therefore, the goal in blood transfusion medicine has primarily been to maximize safety. A number of approaches, including extended testing for transmissible disease markers, treatment of blood products to reduce or inactivate pathogens, and strategies to limit the number of donors to which a recipient is exposed, have been utilized for blood transfusion safety (28).
C. pneumoniae is an obligate intracellular pathogen and infects a variety of cells, including epithelial, endothelial, smooth muscle, macrophages, and lymphocytes (1, 7, 8, 10, 15, 21). A significant repository for this pathogen in blood is circulating leukocytes. In fact, C. pneumoniae can be recovered from CD3-positive peripheral blood leukocytes obtained from patients with cardiovascular disease (11). Our previous study also showed that a significant number of leukocyte samples obtained from healthy donors were demonstrated to have viable C. pneumoniae (29). Therefore, a certain prevalence of resident C. pneumoniae in leukocytes of blood collected for transfusion is very likely. However, it is still unclear whether the resident C. pneumoniae in blood are a potential risk factor in a health issue of the donors. Even though there is a lack of direct evidence on the pathogenesis of resident C. pneumoniae in blood, eradication of the bacteria may be beneficial in blood transfusion. In this regard, whether the eradication of these organisms from blood components by the procedures utilized in practice is possible remains unclear. Therefore, in the present study the practical efficacy of leukoreduction by filtration for depletion of resident C. pneumoniae from red blood cell (RBC) units was examined.
MATERIALS AND METHODS
Blood component source. Whole blood units were obtained from 30 healthy donors (11 males: mean age and standard deviation, 46 ± 14 years; 19 females: 47 ± 19 years) who donated blood at Florida Blood Services, St. Petersburg, Fla. The RBC concentrate was prepared from whole blood donations collected in Baxter triple bags (Baxter, Deerfield, Ill.). The blood was centrifuged at 4,000 rpm for 7 min. Leukocyte reduction was performed up to day 5 of storage.
Leukoreduction. Twelve RBC units were used for the leukoreduction study. The leukoreduction was performed by routine procedures using a leukocyte-depleting filter (Sepacell R-500, Asahi Medical Co., Tokyo, Japan) according to the manufacturer's instructions. Approximately half of the unit's volume was processed for leukoreduction by filtration and the remaining half was used as the prefiltration control.
Detection of C. pneumoniae in RBCs. The presence of C. pneumoniae in RBC units before (prefiltration) and after leukoreduction was assessed by real-time PCR specific for C. pneumoniae 16S rRNA (3). DNA was extracted from 10 ml of RBCs before and after leukoreduction aliquots using QIAmp blood maxi kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. The resulting DNA solutions (1.0 ml) were precipitated with 100 μl 3 M sodium acetate and 2.2 ml ethanol. The precipitates were washed with 70% ethanol and then dissolved in 50 μl AC buffer (QIAGEN); 2 μl of extracted DNA was subjected to real-time PCR.
The DNA was also extracted from the trapped leukocytes on a filter. The filter was washed with Hanks' balanced salt solution. Trapped cells were then eluted from the filter with 10 ml of 8 M urea two times followed by washing with 10 ml of Hanks' balanced salt solution three times. The total eluates were centrifuged for 30 min at 3,615 x g. The resulting precipitates were used for extraction of bacterial DNA using QIAmp DNA mini kit (QIAGEN) with a bacterial DNA extraction protocol. The DNA was dissolved in 50 μl AC buffer.
Real-time PCR was performed in the master mixture (SYBR Green PCR Master Mix; Applied Biosystems, Foster City, Calif.) with primers (sense, 5'-GGA CCT TAG CTG GAC TTG ACA TGT-3'; antisense, 5'-CCA TGC AGC ACC TGT GTA TCT G-3') (3) in an iCycler thermal cycler (Bio-Rad Laboratories, Hercules, Calif.). The thermal cycling conditions were 95.0°C for 10 min and 50 cycles of 95.0°C for 15 s, 63.5°C for 15 s, and 69.5°C for 20 s. The profiles of melting temperatures were assessed for each PCR run for confirmation of the specificity of PCR products. The specificity of amplified target genes was also confirmed in a representative positive sample by direct oligonucleotide sequencing of the PCR products.
As a standard for C. pneumoniae 16S rRNA, a series of diluted C. pneumoniae DNAs extracted from purified C. pneumoniae AR-39 elementary bodies were used (14). The lower detection limit was one inclusion-forming unit per PCR. The relative concentrations of C. pneumoniae DNA (relative number of bacteria) were calculated from the standard curve. Each of the DNA samples was tested by PCR at least three times. To prevent carryover contamination, an aerosol-resistant tip was used in all steps. Preparation of the PCR mixture and PCR were performed in separate rooms.
The immunostaining of C. pneumoniae in eluates of filters was performed with Chlamydia genus-specific fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Research Diagnostics, Flanders, N.J.). The precipitates of eluates were placed on a glass slide, fixed with ethanol, and then stained with FITC-labeled chlamydial antibody, as described previously (15). FITC-labeled mouse immunoglobulin G (BD PharMingen, San Diego, Calif.) was used as a control. The presence of chlamydiae was determined by fluorescence microscopy.
RESULTS
Detection of C. pneumoniae in RBC units. Thirty RBC units were assessed for the presence of C. pneumoniae DNA by real-time PCR specific for C. pneumoniae after extraction of DNA from 10 ml blood sample. Nineteen of 30 units tested showed positive results. The relative bacterial number calculated from the standard curve (correlation coefficient, 0.992; slope, –3.659; intercept, 44.734; 100 to 105 elementary bodies per PCR) of the PCR-positive RBC units was 41.8 ± 50.2 (mean ± standard deviation for 19 samples) per ml blood. The range of bacteria levels was 3 to 179 per ml blood. The profiles of melting temperatures of PCR positive samples showed a single peak identical to the profile of the standard reference C. pneumoniae DNA.
Before and after leukoreduction measures. Twelve of 30 RBC units were randomly selected for leukoreduction study. White blood cell counts after leukoreduction by filtration with filters showed a marked reduction of the leukocyte numbers in filtered blood units (Table 1). However, a minimal number of leukocytes were still detected in the filtered blood units. Eight of 12 RBC units tested were C. pneumoniae positive before leukoreduction. Figure 1 shows the representative PCR results of three RBC units before and after leukoreduction. The profiles of melting temperatures (Fig. 1b) were identical to the reference C. pneumoniae DNA profile. After leukoreduction by filtration, only one unit still showed a PCR-positive result with approximately 10 bacteria per ml of blood. Other RBC units did not show any C. pneumoniae DNA in samples after leukoreduction.
Detection of C. pneumoniae in filters. In order to determine the presence of bacteria in trapped cells by filtration during the leukoreduction, the cells trapped were eluted with urea solution. The presence of C. pneumoniae in the precipitates of the solution by centrifugation was assessed by PCR and immunostaining method. A representative fluorescence microscopic image of samples is shown in Fig. 2. There were several spots stained specifically with FITC-labeled antichlamydia monoclonal antibody in the eluates. Most of the cells observed by microscopy lost cell shape due to treatment with urea. Staining with FITC-labeled mouse immunoglobulin G did not show any spot in these samples (data not shown).
Detection of C. pneumoniae DNA in eluates by PCR also demonstrated that the bacteria were trapped by filters. Table 1 shows the results of PCR detection of the bacteria in the filter eluates. All filters, except one used for filtration of bacteria-positive RBC units, showed PCR as well as immunostaining positivity. Only one filter was not PCR positive even though the RBC unit used showed the presence of the bacteria before filtration by this filter. The levels of bacteria detected in the eluates of the filters were relatively low. Since the filters utilized were composed of multiple membrane layers, elution of all trapped cells was difficult.
DISCUSSION
This study revealed a high prevalence of C. pneumoniae determined by PCR in RBC units for transfusion, even though only a limited number of RBC units were assessed. Since the prevalence of C. pneumoniae in blood specimens determined by PCR may be dependent on several technical factors, such as DNA extraction efficacy and PCR protocols, including selection of target genes and primer sequences (30) as well as target donor subjects, the difference in C. pneumoniae detection rates in blood specimens between published reports which employed different PCR protocols and donor subjects seems reasonable. In fact, the prevalence of C. pneumoniae determined by PCR in the blood of individuals, including patients with cardiovascular diseases, varies from 0 to 49%, depending on the report (3, 4, 6, 16-18, 22, 24, 25). In addition, a small number of C. pneumoniae in blood specimens also contributes to variation in the prevalence due to the low sensitivity of a single PCR determination (26).
In the present study, DNA extraction was performed using a commercial DNA extraction kit with a silica-based membrane which enabled us to purify relatively small amounts of DNA (30). This protocol also handled relatively large amounts of blood specimens (up to 10 ml). The real-time PCR employed in this study, originally reported by Berger et al. (3), detected as few as one organism per PCR using a SYBR Green protocol. Thus, the relatively efficient DNA extraction protocol with a large amount of blood specimens and the sensitive PCR protocol revealed a high prevalence of C. pneumoniae in RBC units.
The number of bacterial cells in positive RBC units varied between 3 and 179 per ml. Since the assessment of bacterial number by PCR may be affected by many factors, as discussed above, the absolute levels of bacteria in RBC units remained to be examined. C. pneumoniae usually results in a persistent infection, which is defined as a long-term association between chlamydiae and the host in which the organisms are viable but in a culture-negative state (2) in infected cells. Therefore, the C. pneumoniae demonstrated in blood components obtained from healthy blood donors without any clinical manifestation are likely to be associated with persistent infection. In addition, the known location of the bacteria in lymphocytes (15, 17) may protect resident C. pneumoniae from host defense mechanisms and be responsible for the lack of clinical manifestations in seemingly healthy blood donors. In this regard, it may be important that assessment of the health condition of the donors demonstrating resident C. pneumoniae in their blood should be followed up for a certain time period after blood donation.
Leukoreduction of blood components by filtration has become common practice. Since resident C. pneumoniae in blood may be located in leukocytes, removal of white blood cells from blood components by filtration may be a practical approach for the reduction of this pathogen. Even though only a limited number of RBC units were examined for assessment of reduction of resident C. pneumoniae by leukoreduction in this study, a marked reduction of bacteria in terms of bacterial number and positivity rate of the bacteria supports the efficacy of the leukoreduction protocol in removing this pathogen from blood units. The effective removal of resident bacteria by filtration was also supported by demonstration of the trapped bacteria in filters by both PCR and immunostaining. The quantitative analysis of the bacteria in eluates obtained from filters did not match the theoretically number of trapped bacteria due to possibly a low elution efficacy of the protocol utilized in this study. Since the filter unit used for leukoreduction is composed of multiple membranes, high yield of bacteria recovery from filters was technically limited. Nevertheless, the recovery of bacteria from the filters used for leukoreduction of bacteria-positive RBC units confirmed the removal of bacteria by filtration.
Even though the bacteria in RBCs after leukoreduction were detected in only one of 12 units tested, the detection of bacteria after leukoreduction indicates that leukoreduction by filtration may not be sufficient for the total eradication of this pathogen in blood components. This conclusion is consistent with the fact that the number of leukocytes, which may be a host cell for C. pneumoniae in blood, was markedly reduced after filtration but a limited number of leukocytes still remained.
ACKNOWLEDGMENTS
This research was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Waksman Foundation Japan Inc.
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Florida Blood Services, St. Petersburg, Florida 33742
Department of Basic Laboratory Sciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
ABSTRACT
Current studies indicate that a significant percentage of healthy blood donors carry Chlamydia pneumoniae in their blood. Although the clinical significance of such findings is unknown, eradication of such bacteria from blood components may contribute to transfusion safety. Deletion of C. pneumoniae in Red Blood Cell (RBC) units was accomplished through leukoreduction by filtration. The presence of bacteria in RBC units before and after leukoreduction was assessed by real-time PCR using primers specific for C. pneumoniae 16S rRNA. The eluates of filters used for leukoreduction were also assessed by PCR and immunostaining with fluorescein isothiocyanate-conjugated chlamydial monoclonal antibodies specific for C. pneumoniae determination. Nineteen of 30 RBC units tested showed the presence of C. pneumoniae DNA. Leukofiltration resulted in a marked reduction of leukocytes as well as C. pneumoniae in terms of bacterial number and positive rate for the bacteria. The eluates of filters showed trapped bacteria determined by both PCR and immunostaining assays. Thus, leukoreduction with a filter is an effective method to significantly reduce resident C. pneumoniae levels in RBC components but may not be completely sufficient for total eradication of this pathogen.
INTRODUCTION
Chlamydia (Chlamydophila) pneumoniae, an obligate intracellular bacterium, causes a variety of respiratory diseases (12). A number of studies indicate that this pathogen is associated not only with respiratory diseases but also with chronic inflammatory diseases, such as atherosclerosis, endocarditis, asthma, and arthritis (9, 13, 20, 23). It has been shown seroepidemiologically that 50 to 80% of the adult population has had prior exposure to this pathogen (5, 11, 27). Although the pathogenic potential of this microorganism in the respiratory system is well established, the pathogen may disseminate systemically from respiratory sites, probably via circulating leukocytes (15, 19). In fact, the presence of C. pneumoniae DNA in the peripheral blood of individuals with cardiovascular diseases as well as healthy subjects has been demonstrated in a significant percentage of these populations (3, 4, 6, 16-18, 22, 24, 25). In addition, another study by us revealed that the bacteria demonstrated in peripheral blood mononuclear cells obtained from healthy blood donors are viable with a readily detectable growth potential (29). Thus, the ubiquitous nature of chronic, persistent infection with C. pneumoniae and the presence of this pathogen in the blood of a potentially significant segment of the blood donor population may be an important consideration for transfusion medicine specialists.
The risk of complications associated with blood transfusion is a major concern to the general public. Therefore, the goal in blood transfusion medicine has primarily been to maximize safety. A number of approaches, including extended testing for transmissible disease markers, treatment of blood products to reduce or inactivate pathogens, and strategies to limit the number of donors to which a recipient is exposed, have been utilized for blood transfusion safety (28).
C. pneumoniae is an obligate intracellular pathogen and infects a variety of cells, including epithelial, endothelial, smooth muscle, macrophages, and lymphocytes (1, 7, 8, 10, 15, 21). A significant repository for this pathogen in blood is circulating leukocytes. In fact, C. pneumoniae can be recovered from CD3-positive peripheral blood leukocytes obtained from patients with cardiovascular disease (11). Our previous study also showed that a significant number of leukocyte samples obtained from healthy donors were demonstrated to have viable C. pneumoniae (29). Therefore, a certain prevalence of resident C. pneumoniae in leukocytes of blood collected for transfusion is very likely. However, it is still unclear whether the resident C. pneumoniae in blood are a potential risk factor in a health issue of the donors. Even though there is a lack of direct evidence on the pathogenesis of resident C. pneumoniae in blood, eradication of the bacteria may be beneficial in blood transfusion. In this regard, whether the eradication of these organisms from blood components by the procedures utilized in practice is possible remains unclear. Therefore, in the present study the practical efficacy of leukoreduction by filtration for depletion of resident C. pneumoniae from red blood cell (RBC) units was examined.
MATERIALS AND METHODS
Blood component source. Whole blood units were obtained from 30 healthy donors (11 males: mean age and standard deviation, 46 ± 14 years; 19 females: 47 ± 19 years) who donated blood at Florida Blood Services, St. Petersburg, Fla. The RBC concentrate was prepared from whole blood donations collected in Baxter triple bags (Baxter, Deerfield, Ill.). The blood was centrifuged at 4,000 rpm for 7 min. Leukocyte reduction was performed up to day 5 of storage.
Leukoreduction. Twelve RBC units were used for the leukoreduction study. The leukoreduction was performed by routine procedures using a leukocyte-depleting filter (Sepacell R-500, Asahi Medical Co., Tokyo, Japan) according to the manufacturer's instructions. Approximately half of the unit's volume was processed for leukoreduction by filtration and the remaining half was used as the prefiltration control.
Detection of C. pneumoniae in RBCs. The presence of C. pneumoniae in RBC units before (prefiltration) and after leukoreduction was assessed by real-time PCR specific for C. pneumoniae 16S rRNA (3). DNA was extracted from 10 ml of RBCs before and after leukoreduction aliquots using QIAmp blood maxi kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. The resulting DNA solutions (1.0 ml) were precipitated with 100 μl 3 M sodium acetate and 2.2 ml ethanol. The precipitates were washed with 70% ethanol and then dissolved in 50 μl AC buffer (QIAGEN); 2 μl of extracted DNA was subjected to real-time PCR.
The DNA was also extracted from the trapped leukocytes on a filter. The filter was washed with Hanks' balanced salt solution. Trapped cells were then eluted from the filter with 10 ml of 8 M urea two times followed by washing with 10 ml of Hanks' balanced salt solution three times. The total eluates were centrifuged for 30 min at 3,615 x g. The resulting precipitates were used for extraction of bacterial DNA using QIAmp DNA mini kit (QIAGEN) with a bacterial DNA extraction protocol. The DNA was dissolved in 50 μl AC buffer.
Real-time PCR was performed in the master mixture (SYBR Green PCR Master Mix; Applied Biosystems, Foster City, Calif.) with primers (sense, 5'-GGA CCT TAG CTG GAC TTG ACA TGT-3'; antisense, 5'-CCA TGC AGC ACC TGT GTA TCT G-3') (3) in an iCycler thermal cycler (Bio-Rad Laboratories, Hercules, Calif.). The thermal cycling conditions were 95.0°C for 10 min and 50 cycles of 95.0°C for 15 s, 63.5°C for 15 s, and 69.5°C for 20 s. The profiles of melting temperatures were assessed for each PCR run for confirmation of the specificity of PCR products. The specificity of amplified target genes was also confirmed in a representative positive sample by direct oligonucleotide sequencing of the PCR products.
As a standard for C. pneumoniae 16S rRNA, a series of diluted C. pneumoniae DNAs extracted from purified C. pneumoniae AR-39 elementary bodies were used (14). The lower detection limit was one inclusion-forming unit per PCR. The relative concentrations of C. pneumoniae DNA (relative number of bacteria) were calculated from the standard curve. Each of the DNA samples was tested by PCR at least three times. To prevent carryover contamination, an aerosol-resistant tip was used in all steps. Preparation of the PCR mixture and PCR were performed in separate rooms.
The immunostaining of C. pneumoniae in eluates of filters was performed with Chlamydia genus-specific fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Research Diagnostics, Flanders, N.J.). The precipitates of eluates were placed on a glass slide, fixed with ethanol, and then stained with FITC-labeled chlamydial antibody, as described previously (15). FITC-labeled mouse immunoglobulin G (BD PharMingen, San Diego, Calif.) was used as a control. The presence of chlamydiae was determined by fluorescence microscopy.
RESULTS
Detection of C. pneumoniae in RBC units. Thirty RBC units were assessed for the presence of C. pneumoniae DNA by real-time PCR specific for C. pneumoniae after extraction of DNA from 10 ml blood sample. Nineteen of 30 units tested showed positive results. The relative bacterial number calculated from the standard curve (correlation coefficient, 0.992; slope, –3.659; intercept, 44.734; 100 to 105 elementary bodies per PCR) of the PCR-positive RBC units was 41.8 ± 50.2 (mean ± standard deviation for 19 samples) per ml blood. The range of bacteria levels was 3 to 179 per ml blood. The profiles of melting temperatures of PCR positive samples showed a single peak identical to the profile of the standard reference C. pneumoniae DNA.
Before and after leukoreduction measures. Twelve of 30 RBC units were randomly selected for leukoreduction study. White blood cell counts after leukoreduction by filtration with filters showed a marked reduction of the leukocyte numbers in filtered blood units (Table 1). However, a minimal number of leukocytes were still detected in the filtered blood units. Eight of 12 RBC units tested were C. pneumoniae positive before leukoreduction. Figure 1 shows the representative PCR results of three RBC units before and after leukoreduction. The profiles of melting temperatures (Fig. 1b) were identical to the reference C. pneumoniae DNA profile. After leukoreduction by filtration, only one unit still showed a PCR-positive result with approximately 10 bacteria per ml of blood. Other RBC units did not show any C. pneumoniae DNA in samples after leukoreduction.
Detection of C. pneumoniae in filters. In order to determine the presence of bacteria in trapped cells by filtration during the leukoreduction, the cells trapped were eluted with urea solution. The presence of C. pneumoniae in the precipitates of the solution by centrifugation was assessed by PCR and immunostaining method. A representative fluorescence microscopic image of samples is shown in Fig. 2. There were several spots stained specifically with FITC-labeled antichlamydia monoclonal antibody in the eluates. Most of the cells observed by microscopy lost cell shape due to treatment with urea. Staining with FITC-labeled mouse immunoglobulin G did not show any spot in these samples (data not shown).
Detection of C. pneumoniae DNA in eluates by PCR also demonstrated that the bacteria were trapped by filters. Table 1 shows the results of PCR detection of the bacteria in the filter eluates. All filters, except one used for filtration of bacteria-positive RBC units, showed PCR as well as immunostaining positivity. Only one filter was not PCR positive even though the RBC unit used showed the presence of the bacteria before filtration by this filter. The levels of bacteria detected in the eluates of the filters were relatively low. Since the filters utilized were composed of multiple membrane layers, elution of all trapped cells was difficult.
DISCUSSION
This study revealed a high prevalence of C. pneumoniae determined by PCR in RBC units for transfusion, even though only a limited number of RBC units were assessed. Since the prevalence of C. pneumoniae in blood specimens determined by PCR may be dependent on several technical factors, such as DNA extraction efficacy and PCR protocols, including selection of target genes and primer sequences (30) as well as target donor subjects, the difference in C. pneumoniae detection rates in blood specimens between published reports which employed different PCR protocols and donor subjects seems reasonable. In fact, the prevalence of C. pneumoniae determined by PCR in the blood of individuals, including patients with cardiovascular diseases, varies from 0 to 49%, depending on the report (3, 4, 6, 16-18, 22, 24, 25). In addition, a small number of C. pneumoniae in blood specimens also contributes to variation in the prevalence due to the low sensitivity of a single PCR determination (26).
In the present study, DNA extraction was performed using a commercial DNA extraction kit with a silica-based membrane which enabled us to purify relatively small amounts of DNA (30). This protocol also handled relatively large amounts of blood specimens (up to 10 ml). The real-time PCR employed in this study, originally reported by Berger et al. (3), detected as few as one organism per PCR using a SYBR Green protocol. Thus, the relatively efficient DNA extraction protocol with a large amount of blood specimens and the sensitive PCR protocol revealed a high prevalence of C. pneumoniae in RBC units.
The number of bacterial cells in positive RBC units varied between 3 and 179 per ml. Since the assessment of bacterial number by PCR may be affected by many factors, as discussed above, the absolute levels of bacteria in RBC units remained to be examined. C. pneumoniae usually results in a persistent infection, which is defined as a long-term association between chlamydiae and the host in which the organisms are viable but in a culture-negative state (2) in infected cells. Therefore, the C. pneumoniae demonstrated in blood components obtained from healthy blood donors without any clinical manifestation are likely to be associated with persistent infection. In addition, the known location of the bacteria in lymphocytes (15, 17) may protect resident C. pneumoniae from host defense mechanisms and be responsible for the lack of clinical manifestations in seemingly healthy blood donors. In this regard, it may be important that assessment of the health condition of the donors demonstrating resident C. pneumoniae in their blood should be followed up for a certain time period after blood donation.
Leukoreduction of blood components by filtration has become common practice. Since resident C. pneumoniae in blood may be located in leukocytes, removal of white blood cells from blood components by filtration may be a practical approach for the reduction of this pathogen. Even though only a limited number of RBC units were examined for assessment of reduction of resident C. pneumoniae by leukoreduction in this study, a marked reduction of bacteria in terms of bacterial number and positivity rate of the bacteria supports the efficacy of the leukoreduction protocol in removing this pathogen from blood units. The effective removal of resident bacteria by filtration was also supported by demonstration of the trapped bacteria in filters by both PCR and immunostaining. The quantitative analysis of the bacteria in eluates obtained from filters did not match the theoretically number of trapped bacteria due to possibly a low elution efficacy of the protocol utilized in this study. Since the filter unit used for leukoreduction is composed of multiple membranes, high yield of bacteria recovery from filters was technically limited. Nevertheless, the recovery of bacteria from the filters used for leukoreduction of bacteria-positive RBC units confirmed the removal of bacteria by filtration.
Even though the bacteria in RBCs after leukoreduction were detected in only one of 12 units tested, the detection of bacteria after leukoreduction indicates that leukoreduction by filtration may not be sufficient for the total eradication of this pathogen in blood components. This conclusion is consistent with the fact that the number of leukocytes, which may be a host cell for C. pneumoniae in blood, was markedly reduced after filtration but a limited number of leukocytes still remained.
ACKNOWLEDGMENTS
This research was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Waksman Foundation Japan Inc.
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