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Qualitative and quantitative detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid from multiple sclerosis patients and controls.

Tang YW, Sriram S, Li H, Yao SY, Meng S, Mitchell WM, Stratton CW - PLoS ONE (2009)

Bottom Line: PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018).None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR.The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America. yiwei.tang@vanderbilt.edu

ABSTRACT
A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

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Dotplot of copy numbers of C. pneumoniae DNA in relapsing remitting MS (left) and progressive MS (right) patients.Horizontal bars indicated the geometric mean copy numbers, which were 567.7 and 1,009.6 copies/ml (geometric mean load) for relapsing remitting and progressive MS, respectively (p>0.05).
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pone-0005200-g001: Dotplot of copy numbers of C. pneumoniae DNA in relapsing remitting MS (left) and progressive MS (right) patients.Horizontal bars indicated the geometric mean copy numbers, which were 567.7 and 1,009.6 copies/ml (geometric mean load) for relapsing remitting and progressive MS, respectively (p>0.05).

Mentions: Quantitative standard curves were achieved by using five 10-fold dilutions of a plasmid standard containing the primer-spanning region of the C. pneumoniae MOMP gene covering plasmid copies from 2.5 to 25,000 per reaction, which corresponded to 25 to 250,000 copies/ml of CSF. A total of 79 CSF specimens were tested to validate the quantitative TaqMan assay. The TaqMan assay detected 26 specimens with a geometric C. pneumoniae mean load equivalent to 774.1 (115.2–6419.8) copies/ml of CSF (Table 3). All 26 positive specimens were from MS patients with positive results by both PCR-EIA and nested-PCR qualitative assays. TaqMan detected 12 (32.4%) of 37 CSF specimens from relapsing remitting MS patients in contrast to 14 (41.2%) of 34 specimens from progressive MS (p>0.05). C. pneumoniae loads measured by TaqMan were higher in CSF from progressive MS (geometric mean load±standard deviation: 1,009.6±3.6 copies/ml) than in CSF from relapsing remitting MS (567.7±3.6 copies/ml), but the difference was not statistically significant (Figure 1). None of the OND controls that were positive or negative by qualitative PCR were positive by TaqMan.


Qualitative and quantitative detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid from multiple sclerosis patients and controls.

Tang YW, Sriram S, Li H, Yao SY, Meng S, Mitchell WM, Stratton CW - PLoS ONE (2009)

Dotplot of copy numbers of C. pneumoniae DNA in relapsing remitting MS (left) and progressive MS (right) patients.Horizontal bars indicated the geometric mean copy numbers, which were 567.7 and 1,009.6 copies/ml (geometric mean load) for relapsing remitting and progressive MS, respectively (p>0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664471&req=5

pone-0005200-g001: Dotplot of copy numbers of C. pneumoniae DNA in relapsing remitting MS (left) and progressive MS (right) patients.Horizontal bars indicated the geometric mean copy numbers, which were 567.7 and 1,009.6 copies/ml (geometric mean load) for relapsing remitting and progressive MS, respectively (p>0.05).
Mentions: Quantitative standard curves were achieved by using five 10-fold dilutions of a plasmid standard containing the primer-spanning region of the C. pneumoniae MOMP gene covering plasmid copies from 2.5 to 25,000 per reaction, which corresponded to 25 to 250,000 copies/ml of CSF. A total of 79 CSF specimens were tested to validate the quantitative TaqMan assay. The TaqMan assay detected 26 specimens with a geometric C. pneumoniae mean load equivalent to 774.1 (115.2–6419.8) copies/ml of CSF (Table 3). All 26 positive specimens were from MS patients with positive results by both PCR-EIA and nested-PCR qualitative assays. TaqMan detected 12 (32.4%) of 37 CSF specimens from relapsing remitting MS patients in contrast to 14 (41.2%) of 34 specimens from progressive MS (p>0.05). C. pneumoniae loads measured by TaqMan were higher in CSF from progressive MS (geometric mean load±standard deviation: 1,009.6±3.6 copies/ml) than in CSF from relapsing remitting MS (567.7±3.6 copies/ml), but the difference was not statistically significant (Figure 1). None of the OND controls that were positive or negative by qualitative PCR were positive by TaqMan.

Bottom Line: PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018).None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR.The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America. yiwei.tang@vanderbilt.edu

ABSTRACT
A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

Show MeSH
Related in: MedlinePlus