A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid
Introduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococc...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
The Journal of Infection in Developing Countries
2016-01-01
|
| Series: | Journal of Infection in Developing Countries |
| Subjects: | |
| Online Access: | https://jidc.org/index.php/journal/article/view/5647 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850186695897513984 |
|---|---|
| author | Idrissa Diawara Khalid Katfy Khalid Zerouali Houria Belabbes Naima Elmdaghri |
| author_facet | Idrissa Diawara Khalid Katfy Khalid Zerouali Houria Belabbes Naima Elmdaghri |
| author_sort | Idrissa Diawara |
| collection | DOAJ |
| description | Introduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis.
Methodology: We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity.
Results: A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4–100) for N. meningitidis and 100% (95% CI = 85.1–100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6–100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods.
Conclusions: Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations.
|
| format | Article |
| id | doaj-art-9724e469fb02423b94fe8a09168e37e5 |
| institution | OA Journals |
| issn | 1972-2680 |
| language | English |
| publishDate | 2016-01-01 |
| publisher | The Journal of Infection in Developing Countries |
| record_format | Article |
| series | Journal of Infection in Developing Countries |
| spelling | doaj-art-9724e469fb02423b94fe8a09168e37e52025-08-20T02:16:18ZengThe Journal of Infection in Developing CountriesJournal of Infection in Developing Countries1972-26802016-01-01100110.3855/jidc.5647A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluidIdrissa Diawara0Khalid Katfy1Khalid Zerouali2Houria Belabbes3Naima Elmdaghri4Faculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, MoroccoUniversity Hospital Centre Ibn Rochd, Casablanca, MoroccoFaculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, MoroccoFaculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, MoroccoFaculty of Medicine and Pharmacy, University Hassan II Ain Chock, Casablanca, MoroccoIntroduction: Acute bacterial meningitis is one of the most severe infectious diseases. Rapid, accurate, and inexpensive diagnosis of bacterial meningitis is crucial for patient management. This study describes a duplex real-time (RT) PCR assay for detection of Neisseria meningitidis and Streptococcus pneumoniae in the cerebrospinal fluid (CSF) for meningitis diagnosis using SYBR Green-based RT-PCR method coupled with melting curve analysis. Methodology: We used SYBR Green-based RT-PCR method coupled with melting curve analysis to detect S. pneumoniae and N. meningitidis in CSF samples. The sensitivity, specificity, and limit of detection were determined. The gold standard for routine tests of CSF analysis is direct examination, culture, and/or latex agglutination. The assay was evaluated on 132 CSF samples to measure clinical sensitivity. Results: A duplex RT-PCR assay for N. meningitidis and S. pneumoniae detection in CSF was evaluated. Two peaks at different melting temperatures (87.5°C and 85.5°C) for N. meningitidis and S. pneumoniae, respectively, were obtained. The sensitivity of RT-PCR was 100% (95% confidence limits [CI] = 82.4–100) for N. meningitidis and 100% (95% CI = 85.1–100) for S. pneumoniae. Specificity was the same (100%) for the bacteria (95% CI = 88.6–100). The percentage of cases accurately diagnosed with meningitis caused by N. meningitidis and S. pneumoniae increased to 50.7% and 28.6%, respectively, when RT-PCR was added to the standard microbiologic methods. Conclusions: Duplex RT-PCR and melting curve analysis with SYBR Green is an inexpensive, sensitive, and specific method to rapidly diagnose bacterial meningitis. Accurate identification of the bacterial causative agents will improve patient management and epidemiological investigations. https://jidc.org/index.php/journal/article/view/5647Neisseria meningitidisStreptococcus pneumoniaemeningitisRT-PCRmelting curve analysis |
| spellingShingle | Idrissa Diawara Khalid Katfy Khalid Zerouali Houria Belabbes Naima Elmdaghri A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid Journal of Infection in Developing Countries Neisseria meningitidis Streptococcus pneumoniae meningitis RT-PCR melting curve analysis |
| title | A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid |
| title_full | A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid |
| title_fullStr | A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid |
| title_full_unstemmed | A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid |
| title_short | A duplex real-time PCR for the detection of Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid |
| title_sort | duplex real time pcr for the detection of streptococcus pneumoniae and neisseria meningitidis in cerebrospinal fluid |
| topic | Neisseria meningitidis Streptococcus pneumoniae meningitis RT-PCR melting curve analysis |
| url | https://jidc.org/index.php/journal/article/view/5647 |
| work_keys_str_mv | AT idrissadiawara aduplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT khalidkatfy aduplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT khalidzerouali aduplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT houriabelabbes aduplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT naimaelmdaghri aduplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT idrissadiawara duplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT khalidkatfy duplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT khalidzerouali duplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT houriabelabbes duplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid AT naimaelmdaghri duplexrealtimepcrforthedetectionofstreptococcuspneumoniaeandneisseriameningitidisincerebrospinalfluid |