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PEDIATRICS Vol. 113 No. 4 April 2004, pp. e318-e321

ELECTRONIC ARTICLE

Is Chlamydia pneumoniae Infection Associated With Stroke in Children With Sickle Cell Disease?

Meenakshi Goyal, MD*, Scott T. Miller, MD*, Margaret R. Hammerschlag, MD{ddagger}, Maureen Gelling, RN, NP{ddagger}, Charlotte A. Gaydos, MS, MPH, DrPH§, Justin Hardick, MS§, Billie Jo Wood, BS§, Tamara Reznik, BS{ddagger} and S.P. Rao, MD*

* Divisions of Hematology-Oncology
{ddagger} Infectious Diseases, Department of Pediatrics, State University of New York, Downstate Medical Center, Brooklyn, New York
§ Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background. Stroke is often a devastating complication of sickle cell disease (SCD). Most children with SCD-related stroke have stenotic and occlusive disease of cerebral blood vessels due to intimal hyperplasia. This hyperplasia is hypothesized to result from an inflammatory response similar to that in atherosclerosis and has been attributed to infection by Chlamydia pneumoniae.

Objective. To determine whether C pneumoniae infection is associated with stroke and cerebrovascular disease, including transient ischemic attacks and abnormal transcranial Doppler examinations, in children with SCD.

Methods. Children with SCD on chronic transfusion due to a history of stroke, transient ischemic attack, or abnormal transcranial Doppler; children with SCD without stroke; healthy controls; and children being transfused for other reasons were enrolled. Peripheral blood and nasopharyngeal (NP) swab specimens were collected from all patients. In patients on transfusion, pretransfusion specimens and samples from the unit of packed red blood cells being transfused were obtained. Peripheral blood monocytic cells (PBMCs) and NP swab specimens were cultured for C pneumoniae in HEp-2 cells. C pneumoniae polymerase chain reaction was performed on PBMCs with a nested touch-down method with primers from the omp-1gene (in duplicate) and a second real-time polymerase chain reaction by using 16S ribosomal RNA primers.

Results. C pneumoniae DNA was detected in the PBMCs of 1 of 14 (7.1%) children with SCD on chronic transfusion, 1 of 10 (10%) sickle cell controls, 1 of 10 (10%) healthy controls, and none of the 5 children receiving chronic transfusion for other reasons. It was not detected in specimens from transfusion units. One child with SCD and stroke, 1 sickle cell control, and 1 transfusion control had positive NP cultures for C pneumoniae. C pneumoniae DNA was not detected in their PBMCs, and all 3 children were asymptomatic. C pneumoniae was not detected by culture of PBMCs from any of the patients after 7 passages.

Conclusion. Stroke in children with SCD does not seem to be associated with C pneumoniae infection in our population.

Key Words: peripheral blood monocytes • transfusion • cerebrovascular disease

Abbreviations: SCD, sickle cell disease • PBMC, peripheral blood monocytic cell • PCR, polymerase chain reaction • NP, nasopharyngeal • LC, Lightcycler • IFU, inclusion-forming unit(s)

Stroke is often a devastating complication that affects 10% of children with sickle cell disease (SCD).1 Most children with sickle cell-related stroke have stenotic and occlusive lesions of large and middle caliber cerebral blood vessels due to intimal hyperplasia.2 This hyperplasia is hypothesized to result from an inflammatory response due to abnormal adhesion of sickle cells to vascular endothelium and is similar to that seen in atherosclerosis.2,3 For atherosclerosis, the exact cause of this inflammation is unknown, but chronic infection has been suggested, with Chlamydia pneumoniae being a likely potential pathogen.3 One of the major limitations of studies of the association of C pneumoniae and atherosclerosis has been the lack of a sensitive and specific test to determine who is actually infected.3 The presence of C pneumoniae DNA in circulating peripheral blood monocytic cells (PBMCs) has been proposed as an indicator of active intravascular infection. C pneumoniae DNA was detected by polymerase chain reaction (PCR) in PBMCs in 0% to 59% of adult patients with coronary artery disease.3,4 However, C pneumoniae DNA was also detected in the PBMCs of 0% to 46% of normal blood donors.3,4

C pneumoniae is a common cause of respiratory infection in adults and children and a frequent cause of acute chest syndrome in children with SCD.5,6 Chronic respiratory infection is known to occur.5 Styles et al7 reported in a preliminary study an association of C pneumoniae infection with sickle cell-related stroke. C pneumoniae DNA was detected in PBMCs of 5 of 10 (50%) children with SCD receiving chronic transfusion for stroke, compared with none of 10 children with SCD and no stroke.

The objective of the present study was to determine whether there indeed is an association of C pneumoniae infection with sickle cell-related stroke. In that the usual therapy for either secondary8 or primary9 stroke prevention in children with SCD is chronic blood transfusion, we also examined whether the presence of C pneumoniae DNA in children with SCD and stroke could be an artifact of transfusion.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Children with SCD with a history of stroke, transient ischemic attacks, or abnormal transcranial Doppler examination9 on chronic transfusion; children with SCD without a history of stroke; patients transfused for other reasons; and hematologically normal children at health maintenance visits were invited to participate in the study. This study was approved by the State University of New York Downstate Medical Center Institutional Review Board. Informed consent was obtained from the parent or legal guardian of each child, and assent was obtained from all children 7 to 18 years old.

Specimen Collection
Nasopharyngeal (NP) specimens for C pneumoniae culture were obtained by using aluminum-shafted Dacron swabs, which were passed through the nares to the posterior nasopharynx. The swabs then were placed into transport media as described.10

Preparation of PBMCs
Peripheral blood was collected by venipuncture for PBMCs by using 8-mL CPT tubes containing sodium citrate (Becton Dickinson, Franklin Lakes, NJ). Blood specimens were stored at room temperature until processing, centrifuged within 2 hours, and processed according to the manufacturer’s package insert. The processed cells were frozen at –70°C until ready for culture and DNA-extraction procedures.

Culture of C pneumoniae
PBMCs and NP specimens were cultured for C pneumoniae in HEp-2 cells grown in 96-well microtiter plates as described.10 Cultures of PBMCs were passaged 7 times.

C pneumoniae PCR
DNA was extracted from 200 µL of processed PBMCs by using the MagnaPur Lightcycler (LC) Robot (Roche Molecular Systems, Pleasanton, CA). Detection of C pneumoniae DNA in PBMCs was performed by using 2 different PCR methods: a nested touch-down PCR with primers from the omp-1 gene (in duplicate)11 and a real-time PCR.12 Real-time PCR was performed by using the existing primer set CPN90 and CPN91; a TaqMan probe was designed for use with CPN90 and CPN91 and the Roche Molecular Systems LC. Primer and probe sequences for the C pneumoniae CPN-LC assay were primer CPN90 (5'-TCTCAACCCCATCCGTGTCGG-3'), primer CPN91 (5'-GCGGAAAGCTGTATTTCTACAGTT-3'), and probe CPNTM (5'-6FAM-ATGCCGCCTGAGGAGTACACTCGCAA-3'). PBMC samples for real-time PCR testing were analyzed under the following conditions: 15 µL of master mix and 5 µL of template DNA with a primer and probe concentration of 10 pm per PCR and a MgCl2 concentration of 4 mM per PCR. The MgCl2, PCR-grade water, and Faststart enzyme were provided by the Roche Molecular Systems Faststart Hybridization Probe Kit. The amount of enzyme (2 µL) was based on manufacturer recommendations. DNA extraction and PCR setup and amplification were all performed in separate rooms. Aerosol barrier-resistant pipette tips were used for all steps in the PCR setup. Cycling conditions were 95°C for 30 seconds of preincubation followed by 45 cycles at 95°C for 0 seconds, 65°C for 10 seconds, and 72°C for 5 seconds, followed by 3 minutes of cooling at 40°C at the end of the cycling protocol. Data collection was performed during extension by the LC software, which calculated cutoff values for positives based on a positive control panel. Negative and positive controls were included in both the extraction and PCR phase of testing. The positive controls consisted of a panel of at least 4 different concentrations of C pneumoniae: 400 inclusion-forming units (IFUs)/PCR, 40 IFU/PCR, 4 IFU/PCR, and 0.4 IFU/PCR.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 39 patients were enrolled, including 13 of 20 children at Downstate Medical Center with sickle cell-related cerebrovascular disease currently on transfusion therapy (7 who had strokes, 2 with recurrent transient ischemic attacks, and 4 with abnormal transcranial Doppler examinations); these children were classified as sickle cell stroke for purposes of analysis. Ten sickle cell control children without a history of stroke, 10 healthy control children, and 5 children receiving chronic transfusion for other reasons (Diamond-Blackfan anemia and thalassemia major) were also enrolled. As shown in Table 1, ages ranged from 7 to 19 years, and 14 patients (35.9%) were male. The mean duration of transfusion therapy in the children with SCD and stroke was 28.22 months (range: 1–72 months), and the median duration was 26 months at the time of study entry.


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  		TABLE 1. Demographic Characteristics of Population

 
C pneumoniae DNA was detected in PBMCs of 1 of 14 (7.1%) children with SCD and stroke and 1 of 10 (10%) non-stroke sickle cell controls, both by only 1 of the 3 replicate PCR tests used (Table 2). C pneumoniae DNA was detected in the PBMCs of 1 of 10 (10%) healthy control children, in this case in all 3 PCR replicates. The prevalence of C pneumoniae DNA was not significantly different between the 3 groups ({chi}2 = 0.1752 with Yates continuity correction; P > .9). C pneumoniae DNA was not detected in the PBMCs of any of the 5 transfusion controls or in specimens from the transfusion units. C pneumoniae was not detected by culture in any subject’s PBMCs. A total of 3 children had positive NP cultures for C pneumoniae: 1 with SCD and stroke, 1 with SCD without stroke, and 1 transfusion control. C pneumoniae DNA was not detected in the PBMCs of any of these children, all of whom were asymptomatic.


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  		TABLE 2. Results of C pneumoniae Cultures and PCR

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cerebrovascular accident is a common complication of SCD, occurring at a rate of ~0.5 events per 100 patient-years of follow-up.1 Of children with sickle cell-related stroke, 70% to 90% have stenotic and occlusive changes in the large- and middle-caliber cerebral blood vessels;2 this vasculopathy has been attributed to damage caused by abnormal adherence of sickle cells13 and leukocytes14 to vascular endothelium, resulting in intimal hyperplasia and narrowing of the vessel lumen.15 Vascular disease is often extensive,16 and stroke recurs in 47% to 90% of untreated children who have an initial stroke.17–19 This risk is reduced greatly by implementation of a chronic transfusion program.8

In the non-sickle cell adult population, similar vascular abnormalities are seen in association with atherosclerosis, and there is considerable interest and controversy as to the role of infection, specifically the role of C pneumoniae infection, in the pathogenesis of these lesions. The rates of identification of C pneumoniae in human atheromas, predominantly by PCR, range from 0% to 100%,3 although the organism has actually been isolated from atheroma tissue in <10% of specimens when culture was performed.3 Inconsistency of results is due, in part, to the lack of standardized methods, especially those using PCR.3,20 Recent studies document major problems with both inter- and intralaboratory reproducibility of in-house PCR assays for C pneumoniae.3,20

It has been suggested that the presence of C pneumoniae in PBMCs may act as a surrogate marker for infection with C pneumoniae in adults with cardiovascular disease.3,4 This is based on the hypothesis that the organism gains access to the vascular system from a respiratory focus through infection of tissue or alveolar macrophages. C pneumoniae has been identified in buffy coats and PBMCs of mice after intranasal inoculation by culture and PCR.21 Similar to the experience with atheroma tissue, the reported prevalence of C pneumoniae DNA in PBMCs has varied significantly, ranging from 0% to 59% of patients with coronary artery disease and from 0% to 46% of healthy blood donors.3,4 Data on the association of the presence of C pneumoniae DNA in PBMCs and atheroma tissue from patients with coronary artery disease in individual studies also have been inconsistent.3 Some of this variation is probably due to variations in PCR methods used, as discussed above. There have been no published studies of the prevalence of C pneumoniae DNA in PBMCs of children. It is also not known whether carriage of C pneumoniae DNA in circulating white blood cells represents a viable infection; actual isolation of viable C pneumoniae from PBMCs by culture is rare.3

Given these methodologic concerns, we felt compelled to attempt to confirm the results reported by Styles et al.7 The results of the present study are strikingly different, which may be due to methodologic issues discussed above. We do not have details of the PCR methods used by Styles et al,7 and they did not perform NP cultures or study a healthy control group or children on chronic transfusion for other reasons.

Our results show no association of infection by C pneumoniae with stroke in children with SCD. There was also no association of presence of C pneumoniae DNA in PBMCs with either NP infection or chronic transfusion. The prevalence of C pneumoniae DNA in these children (7.1%–10%) was similar to that reported in healthy adult blood donors.3,5 In 2 of the children in whom C pneumoniae DNA was detected in PBMCs, only 1 of 3 PCR tests were positive, suggesting a low copy number. Tondella et al22 reported similar results in an adult population. They analyzed 228 PBMC specimens by 4 PCR assays including 2 real-time assays (1 of which was used in the present study), 1 nested PCR assay, and the nested touch-down assay (also used in the present study). One of the real-time assays and the nested PCR assay each detected C pneumoniae DNA in a single but different PBMC specimen, consistent with low amounts of C pneumoniae DNA.

The overall prevalence of asymptomatic C pneumoniae NP infection (7.7%) in the present study was similar to that reported previously in children and adults.23 We conclude that C pneumoniae is not involved in sickle cell-associated cerebrovascular disease.


   FOOTNOTES
 
Received for publication Sep 15, 2003; Accepted Nov 12, 2003.

Reprint requests to (M.R.H.) State University of New York, Downstate Medical Center, Box 49, 450 Clarkson Ave, Brooklyn, NY 11203-2098. E-mail: mhammerschlag{at}pol.net


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PEDIATRICS (ISSN 1098-4275). ©2004 by the American Academy of Pediatrics




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