Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families - detection of carrier status in symptomatic and asymptomatic female relatives.

A comprehensive study of the Hungarian Duchenne/Becker muscular dystrophy (DMD/BMD) families is presented. Deletions in the hot spots regions were identified by multiplex PCR, whereas rare mutations were detected by Southern blot and multiplex ligation-dependent probe amplification (MLPA) techniques. DMD/BMD disease was confirmed and exact deletion borders were determined in 19 out of 135 affected males using multiplex PCR. Additional exons involved as well as rare exon deletions were identified by MLPA in 71 male patients, whereas duplications were observed in seven patients. In two DMD patients, the entire dystrophin gene and adjacent genes were deleted. Out of the 95 female relatives, 41 proved to be carriers, including three manifesting carrier females. Using MLPA method, a large portion of the Hungarian DMD/BMD patients and their female relatives were exactly genotyped. For the first time, the incidence and prevalence of asymptomatic and symptomatic female carriers in Hungary was estimated.

Under-expression of CD24 in pre-eclamptic placental tissues determined by quantitative real-time RT-PCR.

BACKGROUND: Pre-eclampsia is a pregnancy-related disorder present in about 5-7% of all pregnancies. CD24 expression was recently reported in different diseases, while it has not yet been determined in pre-eclamptic placental tissues. METHODS: We collected placental tissues from pre-eclamptic (n = 16) and healthy pregnancies (n = 16). We used the quantitative real-time PCR method with a primer-probe system for determination of CD24 gene expression. RESULTS: We measured CD24 concentrations of 18.94 +/- 26.86 ng/microl in the pre-eclamptic and 53.85 +/- 92.05 ng/microl in the healthy placental tissues (p = 0.03). CONCLUSIONS: The quantitative real-time PCR method is suitable to determine CD24 expression in placental tissues. We suppose the low expression of CD24 may cause the enhanced immune reaction and could play a role in the abnormal development of placenta in pre-eclampsia.

[Carrier detection in families affected by Duchenne/Becker muscular dystrophy]. / Hordozóságszurés Duchenne-/Becker-izomdystrophiával érintett családokban.

Duchenne/Becker muscular dystrophy is a severe, recessive, X-linked neuromuscular disease with an incidence of 1/3500 (Duchenne type) and 1/30,000 (Becker type) in newborn boys. The gene responsible for the Duchenne/Becker muscular dystrophy phenotype is located at Xp21 and its 427 kD protein product is called dystrophin. Deletions, point mutations and rarely duplications can occur almost anywhere in the DMD gene, which makes the molecular diagnosis difficult. Multiple polymerase chain reactions detect 95% of deletions in affected males [2, 4], but are not suitable for carrier detection in female relatives. Southern-blot analysis with six different cDNA probes covers the whole 14 kb dystrophin transcript and allows the detection of female carriers by comparing the intensity of the signals corresponding to the different exons. This method is time consuming compared to the newly introduced multiple ligation-dependent probe amplification method. Multiple ligation-dependent probe amplification is a method suitable for relative quantification of several DNA sequences in one reaction. The authors report results on 93 cases where the carrier status was analysed simultaneously by cDNA hybridisation and multiple ligation-dependent probe amplification technique. In 42 cases the carrier state was confirmed and in this carrier population the authors additionally detected two cases with duplication, two cases with one copy of the whole dystrophin gene and three manifest carrier females. On the basis of these results the MLPA technique, which has been newly introduced in Hungary, proved to be a sensitive and quick method for the detection of carrier state in the DMD/BMD disease. Moreover, the exact deletion or duplication border can be detected and as a result, prediction on the phenotype can be given. This will provide the right therapeutic intervention for the affected patients in the future.

[Detection of Toxoplasma gondii in amniotic fluid using quantitative real-time PCR method]. / Toxoplasma gondii kimutatása magzatvízbol kvantitatív valós ideju PCR-módszerrel.

INTRODUCTION: The infection caused by parasite Toxoplasma gondii is often asymptomatic or a mild clinical disease. Congenital toxoplasmosis is the result of transplacental transmission of Toxoplasma gondii from an acute infected mother. Toxoplasmosis can cause several fetal symptoms. Early diagnosis of the infection can enhance the success of the medical treatment. Congenital toxoplasmosis can be detected by serological or PCR amplification methods. AIMS: The authors decided to develop a quantitative real-time PCR technique for detection of T. gondii in amniotic fluid samples. METHODS: DNA was isolated using silica adsorption method. Quantitative real-time PCR method was used to detect T. gondii infection in the samples. RESULTS: From the studied 74 samples in 6 cases T. gondii was detected. CONCLUSION: The introduced quantitative real-time PCR method is a fast and sensitive PCR based method and makes possible the quantification of the protozoa number in the sample.

[Non invasive detection of fetal Rh using real-time PCR method]. / Noninvazív magzati Rh-meghatározás real-time PCR-módszerrel.

INTRODUCTION: In the last ten years the detection of fetal origin cells and cell free fetal DNA in maternal circulation opened new horizons in non-invasive prenatal diagnosis. The diagnostic possibilities are based on the differences between the maternal and fetal origin DNA. One of the differences could be the Rh blood group and the genetical background. The Rh incompatibility is the most frequent blood group incompatibilities in the clinical practice, which can cause fetal anemia, hydrops and even fetal death. AIMS: The aim of this study was to detect the fetal DNA in maternal circulation, to determine the Rh status of the fetus, and to compare the reliability of the method with the data found in other studies. METHODS: Blood samples and amnionic fluid samples were collected from 30 pregnant women, with Rh negative status, between 11-22 week of gestation presented for genetic amniocentesis at the 1st. Department of Obstetrics and Gynecology, Semmelweis University. After DNA isolation real-time PCR was performed in order to detect the exon 7 of the RhD gene located on the first chromosome (1p36.11.). RESULTS: In 24 cases the PCR reaction gave same result in case of the DNA isolated from plasma and amniotic fluid, but in six cases there was no PCR product of plasma samples and the product was detectable in amniotic fluid samples. The exon 7 was detectable in 25 cases, and there was no product in 5 cases. CONCLUSIONS: The real-time PCR method seems to be an easy and reliable method to determine the fetal Rh blood group. The sensitivity and specificity of the method in this study is in concordance with international data. The use of more than one probe could increase the sensitivity of the method.

Role of second trimester sonography in detecting trisomy 18: a review of 70 cases.

PURPOSE: To investigate the role of second-trimester sonographic examination in the prenatal diagnosis of trisomy 18. METHODS: Out of 22,150 fetal chromosomal analyses performed between 1990 and 2004, 70 trisomy 18 fetuses were found. The sonographic findings of this aneuploidy were analyzed. RESULTS: The average maternal age was 32.4 years; the average gestational age was 19.5 weeks. Major anomalies were seen in 61 (87.1%) of the 70 fetuses with trisomy 18; among these, cardiac anomalies were the most common (47.1%), with a 27.1% incidence of ventricular septal defects. Anomalies of the central nervous system were seen in 35.7% of cases; abnormal head shape was the most frequently detected anomaly in this group (12.9%). Fifty-six (80%) of the fetuses had at least 1 minor anomaly; of these, choroid plexus cyst was the most common (38.6%). Increased nuchal fold thickness was detected in 17.1% of cases. CONCLUSION: The vast majority of trisomy 18 fetuses have sonographically detectable abnormalities in the second trimester. Both the 87.1% frequency of major anomalies and the 80% frequency of minor anomalies are substantially higher than multiple biochemical marker tests could achieve. It was also demonstrated that fetal echocardiography plays a pivotal role in the diagnosis of trisomy 18.

Detection of DeltaF508del using quantitative real-time PCR, comparison of the results obtained by fluorescent PCR.

OBJECTIVE: Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in the Caucasian population. The molecular diagnosis is difficult since there are about 1,000 mutations in the CF transmembrane regulator gene. The DeltaF508del is the cause of the CF in 64% of the cases in Hungary. Our aim was to compare two polymerase chain reaction (PCR)-based method for the detection of DeltaF508del. METHODS: One hundred and sixteen DNA samples isolated from different tissues (84 blood samples, 18 chorionic villus samples and 14 amniotic fluid samples) were involved in the study. Fluorescent PCR with DNA fragment analysis and quantitative real-time PCR with melting curve analysis were performed on the DNA samples for the detection of DeltaF508del. RESULTS: Sixty-five healthy normal samples, 43 heterozygous samples, 6 DeltaF508del homozygous samples and 2 DeltaF508C homozygous samples were detected by using quantitative real-time PCR combined with melting curve analysis. The fluorescent PCR method did not detect the DeltaF508C mutation and these two samples were diagnosed as normal healthy ones. CONCLUSIONS: The quantitative real-time PCR with melting curve analysis is a reliable and fast method for the detection of DeltaF508del. The results are ready in 1 h following the DNA isolation. The applied primer-probe set with melting curve analysis gives additional information for the presence of other mutations in the DeltaF508del region.

Prenatal sonographic findings in 207 fetuses with trisomy 21.

OBJECTIVE: The objective was to evaluate the contribution of second trimester ultrasound examination to the prenatal diagnosis of trisomy 21 in 207 fetuses with this aneuploidy. The type and frequency of abnormal sonographic findings were determined. Possible multiple malformation patterns, characteristic of trisomy 21 were sought. STUDY DESIGN: Singleton fetuses that had prenatal sonography during the second trimester, then underwent cytogenetic evaluation in our institution, made up the study population. The sonographic findings of 207 fetuses with trisomy 21 were analyzed. RESULTS: Between 1990 and 2004, fetal karyotyping was performed in 22,150 patients for different indications. An abnormal karyotype was diagnosed in 514 cases (2.3%); among them 207 fetuses with trisomy 21 were detected (40.3%). Abnormal sonography was seen in 63.8% of the cases. Structural anomalies were detected in 28.5% of the trisomy 21 fetuses, among them cardiac defects (15.9%), central nervous system anomalies (14.5%), and cystic hygromas (6.8%) were the most common. Of the minor markers, increased nuchal translucency (28%), pyelectasis (20.3%), and shorter extremities (8.7%) were common findings. CONCLUSIONS: Appropriate diagnosis of structural anomalies, looking for relatively easily detectable minor markers and incorporating fetal echocardiography into the second trimester sonographic protocol, may increase the contribution of mid-trimester ultrasound examination to diagnosing trisomy 21.

[Detection of deltaF508 deletion causing cystic fibrosis, using quantitative real-time PCR]. / A cystis fibrosist okozó F508 deléció kimutatása kvantitatív real-time PCR-módszerrel.

INTRODUCTION: Cystic fibrosis is the most common autosomal recessive lethal genetic disorder in the Caucasian population. There are about 1400 mutation in the cystic fibrosis transmembrane regulator gene, which makes the molecular diagnosis difficult, while luckily in Hungary the cause is the deltaF508del in almost 60% of the cases. METHOD: The authors introduced the quantitative real-time PCR and melting curve analysis method for the detection of deltaF508del. They studied 94 samples (70 blood, 16 chorionic villi, 8 amniotic fluids). RESULTS: They found 52 healthy normal, 36 heterozygotic and 5 homozygotic samples and one deltaF508C homozygotic sample. DISCUSSION: The quantitative real-time PCR and melting curve analysis is a reliable and fast method for detection of deltaF508del. The results are available in one hour following the DNA isolation. The primer-probe set makes available the deltaF508Cdel detection too.

Presence of cell-free fetal DNA in plasma of women with ectopic pregnancies.

BACKGROUND: The quantity of cell-free fetal DNA in the plasma of pregnant women changes during pregnancy and seems to be different in normal and pathologic pregnancies. We investigated the possible diagnostic applications of the detection and measurement of cell-free fetal DNA by comparing quantities found in women with ectopic (EP) or intrauterine (IUP) pregnancies. METHODS: We collected blood samples from 58 women who had positive pregnancy tests and specific complaints and sonographic findings suggestive of EP and from 45 women with confirmed IUP. We performed quantitative real-time PCR analysis of the sex-determining region Y (SRY) gene to detect and measure the amount of cell-free fetal DNA. The diagnosis of EP was confirmed by histologic examination. RESULTS: SRY was detected in 15 EP and 14 IUP cases. The mean (SD) amount of cell-free fetal DNA was significantly higher (P<0.005) in women with EP [565 (136) genome-equivalents (GE)/mL] than in women with IUP [72 (19) GE/mL] at the same gestational age. CONCLUSIONS: Our results confirm that cell-free fetal DNA is present in plasma of women with EP. The finding of higher amounts of cell-free fetal DNA in EP cases than in IUP cases suggests that this method might be useful for early diagnosis of EP.

Prenatal diagnosis of Turner syndrome: report on 69 cases.

OBJECTIVE: This study was conducted to evaluate the diagnostic value of different sonographic signs of fetuses with Turner syndrome in the first and second trimesters of pregnancy. METHODS: Between 1990 and 2004, Turner syndrome was found in 69 of 22,150 fetal karyotypings. Congenital anomalies detected by sonography were analyzed. RESULTS: Of the 514 (2.3%; 514/22,150) chromosome aberrations that were diagnosed, 69 Turner syndrome cases were found (13.4%; 69/514). Twenty-four fetuses had a 45,X karyotype (34.8%), and 45 fetuses were mosaic (65.2%). Forty-seven fetuses (68.1%; 47/69) showed symptoms on sonography. A substantial proportion of fetuses with Turner syndrome showed early-onset signs that could be detected in the first trimester (29.8%;14/69). The most common findings with sonography were hygroma colli (26.1%; 18/69), fetal hydrops (11.6%; 8/69), cardiac defects (13%; 9/69), and increased nuchal translucency (13%; 9/69). Among heart defects, coarctation of the aorta was the most common (44.4% of all cardial defects). Soft markers were also detected with relatively high frequency (23.2%; 16/69). CONCLUSIONS: The diagnosis of severe Turner syndrome is possible in early pregnancy. A search for soft markers during second-trimester sonography and extensive use of echocardiography may increase the detection rate of Turner syndrome.

Prenatal diagnosis of trisomy 13: analysis of 28 cases.

OBJECTIVE: The purpose of this study was to investigate the role of second-trimester sonographic examination in the prenatal diagnosis of trisomy 13. METHODS: Of 22,150 fetal chromosome analyses, 28 fetuses with trisomy 13 were found between 1990 and 2004. Sonographic findings of this aneuploidy were analyzed in this study. RESULTS: The average maternal age was 32.4 years; the average gestational age was 19.5 weeks. There was an 89.3% (n = 25) total prevalence of sonographic abnormalities in fetuses with trisomy 13 in this series. Major (structural) malformations were seen in 23 cases (82.1%), whereas minor anomalies were detected on sonography in 16 cases (57.1%). Although in 2 fetuses 1 minor anomaly was the only sonographic sign of trisomy 13, other cases with minor anomalies (87.5% [n = 14]) were multiplex malformations, in which combinations of major and minor anomalies were detected on sonography. The most frequently seen structural abnormalities were central nervous system and facial anomalies (64.3% [n = 18]). Among central nervous system anomalies, ventriculomegaly and holoprosencephaly were seen most frequently. Cardiovascular anomalies were detected in 53.6% (n = 15) of the fetuses with trisomy 13. This high frequency underlines the importance of echocardiography in diagnosing this aneuploidy. Among minor anomalies, increased nuchal translucency (21.4%) and echogenic bowel (17.9%) were the most common findings. CONCLUSIONS: Second-trimester sonographic examination is capable of showing anomalies that are characteristic of trisomy 13; thus, the scan can indicate whether fetal karyotyping is advisable. Incorporation of careful assessment of the fetal cardiovascular system by sonography certainly increases the detection rate of trisomy 13.

Detection of maternal deoxyribonucleic acid in peripheral blood of premature and mature newborn infants.

BACKGROUND: Over the past decade, a lot of attention has been directed towards the fetomaternal and maternofetal transfer of nucleated cells and plasma DNA. In some autoimmune diseases, the fetal DNA is suspected to play an important role in the etiology of the disease. In the same way, the presence of maternal cells and free plasma DNA in fetal/newborn circulation gives rise to interesting questions. The aim of our study was to detect maternal deoxyribonucleic acid in the peripheral blood of premature and mature newborn infants. METHODS: In the case of eight RhD-positive mothers-RhD-negative newborn pairs, peripheral blood samples were collected from the newborn infants within 35-120 min after birth. The maternal origin DNA was determined by real-time PCR amplification of the exon 7 of the RhD-positive allele. RESULT: In all eight cases, the RhD exon 7 was amplified during the PCR reaction. CONCLUSION: The result of our study demonstrates that maternal DNA is present in the fetal peripheral circulation. The presence of maternally derived cells/DNA in the blood of newborn infants might have a role in the immunization of the newborn infants and also could be a possible explanation for 'grandmother effect' in the case of Rh-negative nulligravida patients.

Detection of Toxoplasma gondii from amniotic fluid, a comparison of four different molecular biological methods.

BACKGROUND: The infection caused by the parasite Toxoplasma gondii (T. gondii) is often asymptomatic or has mild symptoms. The infection can cause serious problems in pregnant women who acquire the infection during gestation and their fetuses are congenitally infected. METHODS: We tested 64 amniotic fluid samples for the presence of T. gondii by using fluorescent PCR and DNA fragment analysis. Later we compared four different molecular biological methods for the detection of the presence of T. gondii on same frozen DNA samples. These methods are the conventional PCR, fluorescent PCR with DNA fragment analysis, quantitative real-time PCR with SYBRGreen I and with fluorescence energy transfer hybridization probe detection. We determined the detection limit of these methods. RESULTS: The conventional PCR and quantitative real-time PCR with SYBRGreen I detection have the detection limit of 1000 parasites, followed by fluorescent PCR with the detection limit of 10-100 parasites. The real-time PCR using fluorescence energy transfer hybridization probes can detect one parasite. This is the most sensitive and the fastest method. We detected 5 T. gondii positive samples with all methods from the studied 64 amniotic fluids. CONCLUSIONS: All studied molecular biological methods are suitable for the detection of congenital toxoplasmosis. The quantitative real-time PCR based methods are more sensitive, simple and easy to perform these are opening the avenue to find out the effect of the number of parasites on fetal abnormalities.

Rapid determination of trisomy 21 from amniotic fluid cells using single-nucleotide polymorphic loci.

OBJECTIVES: Rapid detection of trisomy 21 is an important goal for prenatal genetic centers. Fluorescent-PCR and DNA fragment analysis was developed a decade ago and thousands of samples were analyzed in routine practice using this method. Quantitative real-time PCR with melting curve analysis using SNP markers for trisomy 21 detection was described recently. We studied the reliability of this method on a cohort of samples of Hungarian patients. METHODS: DNA was isolated with silica adsorption method from amniotic fluid cells. We investigated 67 trisomy 21 and 62 diploid samples in the study. Quantitative real-time PCR was performed using hybridization probes combined with melting curve analysis. Peak areas under the derivative curves were determined and analyzed. RESULTS: The SNP marker WIAF 899 was informative in 41.86% of cases and WIAF 2643 in 48.83%. The melting curve area ratios were significantly different between trisomic and normal cases for WIAF 899 (trisomic 0.5246 +/- 0.2498 vs 0.8347 +/- 0.5234; p < 0.001), while in the case of WIAF 2643, they were not different. CONCLUSION: Combined and selected SNP markers could be valuable tools for rapid trisomy 21 detection in prenatal genetic screening.

[Effect of the DNA isolation method on the results of multiple fluorescent PCR and DNA fragment analysis]. / A DNS-izolálási módszer hatá'sa a multiplex fluoreszcens PCR- es DNS-fragmens-analízis eredményeire.

INTRODUCTION: The quality of the nucleic acids has tremendous effects on the results of molecular diagnostic tests. METHODS: The authors have isolated DNA from amniotic fluid samples by using rezin-binding and silica adsorption methods. In the case of the rezin-binding technique they used both frozen and fresh samples. The quality of the isolated DNA was compared by fluorescent PCR and DNA fragment analysis. RESULTS: With the use of the rezin-binding method on frozen samples the authors obtained more PCR fragments following the fluorescent PCR amplification. The gender and the aneuploidy screening is easier from the electrophoretogramms using better quality DNA. Out of the three studied DNA isolation method the silica adsorption seemed to be the best for the fluorescent-PCR and DNA fragment analysis. DISCUSSION: The authors found the silica adsorption method is more suitable for DNA isolation from amniotic fluids for fluorescent PCR and DNA fragment analysis amplification. They suppose silica adsorption DNA isolation results in a total removal of the PCR inhibitors.

The DNA isolation method has effect on allele drop-out and on the results of fluorescent PCR and DNA fragment analysis.

BACKGROUND: The quality and the quantity of isolated DNA have an effect on PCR amplifications. METHODS: The authors studied three DNA isolation protocols (resin binding method using fresh and frozen amniotic fluid samples, and silica adsorption method using fresh samples) on the quantity and on the quality of the isolated DNA. Amniotic fluid samples were obtained from 20 pregnant women. The isolated DNA concentrations were determined by real-time fluorimeter using SYBRGreen I method. Each sample was studied for the presence of 8 STR markers. The authors compared the number of the detected alleles, electrophoretograms and peak areas. RESULTS: There was a significant difference between the concentration of the obtained DNA and in the peak areas between the three isolation protocols. The numbers of detected alleles were different, we observed the most allele drop outs in the resin type DNA isolation protocol from the fresh sample (detected allele numbers 182), followed by resin binding protocol from the frozen samples (detected allele number 243) and by the silica adsorption method (detected allele number 264). CONCLUSIONS: The authors demonstrated that the DNA isolation method has an effect on the quantity and quality of the isolated DNA, and on further PCR amplifications.

Isolation of epsilon-haemoglobin-chain positive fetal cells with micromanipulation for prenatal diagnosis.

OBJECTIVES: The isolation and analysis of fetal cells in maternal blood during pregnancy is under investigation as a means of noninvasive prenatal diagnosis. The aim of our study was to detect fetal gender from maternal peripherial blood samples during pregnancy with the detection and analysis of epsilon-haemoglobin-chain positive fetal nucleated red blood cells (NRBCs) collected by a micromanipulator. Here we report our first results. DESIGN AND METHODS: We obtained maternal blood from 14 singleton pregnancies. After a double density gradient separation, magnetic activated cell sorting was performed by positive selection for nucleated red blood cells with anti-CD71. With the help of this enrichment step, followed by immunophenotyping with an anti-haemoglobin-epsilon monoclonal antibody, the isolation of the epsilon haemoglobin chain positive cells with micromanipulation could be done. We performed single cell fluorescent PCR analysis of these cells; we used primers for the amelogenin gene to detect fetal gender. We compared our findings with the results of amniocentesis. RESULTS: Fetal gender was successfully determined in 11 out of 14 cases; among them, in 2 cases with Klinefelter syndrome (47,XXY). CONCLUSION: The results of our study suggest that micromanipulation and QF-PCR analysis of anti-haemoglobin-epsilon fluorescent antibody stained fetal cells from maternal blood can be useful in prenatal diagnosis to detect fetal gender and promising to be improved to detect chromosomal abnormalities.

Prenatal diagnosis, phenotypic and obstetric characteristics of holoprosencephaly.

The diagnosis of fetal malformations, especially those of the central nervous system, is strikingly important in the practice of genetic counseling. Early diagnosis is very significant, not only because of the prognosis, but also because of the emotional effects caused by the accompanying craniofacial malformations. The summary of the obstetrical and diagnostical characteristics should be useful in the management of holoprosencephaly. The analysis of the 50 cases we encountered between 1981 and 2000, including the anatomical, diagnostic and clinical aspects, as well as the associated craniofacial malformations, forms the essence of our publication. In one of the examined cases a familiar recurrence was verified.

Chlamydia pneumoniae in coronary bypass grafts of redo patients. The concept of the 'adventitial baseline infection'.

The pathogenic role of Chlamydia pneumoniae in late coronary bypass graft failure has not yet been extensively investigated. We examined failed and new arterial/venous bypass grafts using immunohistochemistry, polymerase chain reaction (PCR), and serology. Thirty-four long-term failed grafts and 28 new grafts were examined in 21 patients undergoing redo coronary artery bypass grafting (CABG). Immunohistochemically, 28 (82%) failed grafts were positive in the intimal-medial compartment, and 33 grafts (97%) were positive for C. pneumoniae in the adventitia. Thirteen (46%) and 27 (96%) new grafts showed infection in the intima-media and in the adventitia, respectively (p < 0.05). Immunohistochemically, the overall presence of C. pneumoniae in all vessels examined was 66% in the intima-media and 97% in the adventitia (p < 0.05). C. pneumoniae was detected by PCR in 19 (31%) of all the vessels examined. C. pneumoniae seems to be frequently present in grafts of patients considered for redo CABG in Hungary. The adventitia of both failed, and new grafts particularly often contained C. pneumoniae. The results suggest that there exists an adventitial baseline infection from which infection of the inner wall layers develops, depending on local microenvironmental conditions. This is the first study to evaluate chlamydial infection in arterial/venous coronary grafts by immunohistochemistry, PCR, and serology.