sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (Ct) value of 35, with 100% average reaction efficiency and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average Ct values from 13.0 to 29.5. The mean sodC Ct value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.

[1]  R. Borrow,et al.  Simultaneous Detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR , 2001, Journal of Clinical Microbiology.

[2]  George M. Carlone,et al.  Evaluation of a Medium (STGG) for Transport and Optimal Recovery of Streptococcus pneumoniae from Nasopharyngeal Secretions Collected during Field Studies , 2001, Journal of Clinical Microbiology.

[3]  P. Cieslak,et al.  Capsule switching of Neisseria meningitidis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  R. Borrow,et al.  Genetic analysis of capsular status of meningococcal carrier isolates , 2003, Epidemiology and Infection.

[5]  H. Fredlund,et al.  Real‐time PCR detection of five prevalent bacteria causing acute meningitis , 2009, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[6]  David L. Wheeler,et al.  GenBank , 2015, Nucleic Acids Res..

[7]  P. Langford,et al.  Presence of Copper- and Zinc-Containing Superoxide Dismutase in Commensal Haemophilus haemolyticus Isolates Can Be Used as a Marker To Discriminate Them from Nontypeable H. influenzae Isolates , 2006, Journal of Clinical Microbiology.

[8]  J. Jordens,et al.  Detection of Meningococcal Carriage by Culture and PCR of Throat Swabs and Mouth Gargles , 2002, Journal of Clinical Microbiology.

[9]  R. Naclerio,et al.  Mediator release after nasal airway challenge with allergen. , 2015, The American review of respiratory disease.

[10]  S. Salzberg,et al.  Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. , 2000, Science.

[11]  J. Sejvar,et al.  Etiologies of bacterial meningitis in Bangladesh: results from a hospital-based study. , 2009, The American journal of tropical medicine and hygiene.

[12]  P. Langford,et al.  Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[13]  E. Bingen,et al.  Bacterial counts in cerebrospinal fluid of children with meningitis , 1990, European Journal of Clinical Microbiology and Infectious Diseases.

[14]  M. Maiden,et al.  Many carried meningococci lack the genes required for capsule synthesis and transport. , 2002, Microbiology.

[15]  Lennart Sjöberg,et al.  Ahlstrand E , Persson L , Tidefelt U , Söderquist B. Alteration of the colonization pattern of coagulase-negative staphylococci in patients undergoing treatment for hematological malignancy. Eur J Clin Microbiol Infect Dis , 2011 .

[16]  J. Møller,et al.  Combined assay for two-hour identification of Streptococcus pneumoniae and Neisseria meningitidis and concomitant detection of 16S ribosomal DNA in cerebrospinal fluid by real-time PCR , 2008, Scandinavian journal of infectious diseases.

[17]  P. Langford,et al.  Copper-zinc superoxide dismutase in Haemophilus species. , 1992, Journal of general microbiology.

[18]  M. Achtman,et al.  Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. van der Ende,et al.  Comparison of commercial diagnostic tests for identification of serogroup antigens of Neisseria meningitidis , 1995, Journal of clinical microbiology.

[20]  Xin Wang,et al.  Meningococcus genome informatics platform: a system for analyzing multilocus sequence typing data , 2009, Nucleic Acids Res..

[21]  M. Taha,et al.  Simultaneous Approach for Nonculture PCR-Based Identification and Serogroup Prediction of Neisseria meningitidis , 2000, Journal of Clinical Microbiology.

[22]  A. McEwan,et al.  Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity , 2001, Molecular microbiology.

[23]  D. Stephens,et al.  Genetic basis for nongroupable Neisseria meningitidis. , 2003, The Journal of infectious diseases.

[24]  J. Lemeland,et al.  Detection of Neisseria meningitidis DNA from skin lesion biopsy using real-time PCR: usefulness in the aetiological diagnosis of purpura fulminans , 2007, Intensive Care Medicine.

[25]  T. Cherian,et al.  Pediatric bacterial meningitis surveillance - African region, 2002--2008. , 2009, MMWR. Morbidity and mortality weekly report.

[26]  K. Sunakawa,et al.  Rapid detection of eight causative pathogens for the diagnosis of bacterial meningitis by real-time PCR , 2009, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy.

[27]  V. Sambri,et al.  Multiple Nucleotide Substitutions in the Neisseria meningitidis Serogroup C ctrA Gene Cause False-Negative Detection by Real-Time PCR , 2010, Journal of Clinical Microbiology.

[28]  W. Verstrepen,et al.  Evaluation of a real-time polymerase chain reaction assay for the diagnosis of pneumococcal and meningococcal meningitis in a tertiary care hospital , 2007, European Journal of Clinical Microbiology & Infectious Diseases.

[29]  A. Chan,et al.  A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies , 2008, The Journal of experimental medicine.

[30]  L. J. La Scolea,et al.  Quantitation of bacteria in cerebrospinal fluid and blood of children with meningitis and its diagnostic significance , 1984, Journal of clinical microbiology.

[31]  T. Popović,et al.  Genotype-specific carriage of Neisseria meningitidis in Georgia counties with hyper- and hyposporadic rates of meningococcal disease. , 2002, The Journal of infectious diseases.

[32]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[33]  T. Popović,et al.  Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae , 1998 .

[34]  R Staden,et al.  The staden sequence analysis package , 1996, Molecular biotechnology.

[35]  Raydel D. Mair,et al.  Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae. , 2011, International journal of medical microbiology : IJMM.

[36]  K. O'Brien,et al.  Report from a WHO working group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae , 2003, The Pediatric infectious disease journal.

[37]  S. Clarke,et al.  Interlaboratory Comparison of PCR-Based Identification and Genogrouping of Neisseria meningitidis , 2005, Journal of Clinical Microbiology.

[38]  Tanja Popovic,et al.  Use of Real-Time PCR To Resolve Slide Agglutination Discrepancies in Serogroup Identification of Neisseria meningitidis , 2004, Journal of Clinical Microbiology.

[39]  J. Gilsdorf,et al.  Prevalence of the sodC Gene in Nontypeable Haemophilus influenzae and Haemophilus haemolyticus by Microarray-Based Hybridization , 2009, Journal of Clinical Microbiology.

[40]  Ellen Jo Baron,et al.  Manual of clinical microbiology , 1975 .

[41]  J. Jordens,et al.  A novel porA-based real-time PCR for detection of meningococcal carriage. , 2005, Journal of medical microbiology.

[42]  A. Steigerwalt,et al.  Evaluation and Improvement of Real-Time PCR Assays Targeting lytA, ply, and psaA Genes for Detection of Pneumococcal DNA , 2007, Journal of Clinical Microbiology.

[43]  Henry D. Isenberg,et al.  Manual of Clinical Microbiology , 1991 .

[44]  P. Langford,et al.  Periplasmic Superoxide Dismutase in Meningococcal Pathogenicity , 1998, Infection and Immunity.