LETTER
The genetic diversity of Neisseria meningitidis makes reliable laboratory detection of this important pathogen difficult. Although matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been widely adopted for identification of bacteria from clinical samples (1–3), it does not yet reliably distinguish between N. meningitidis and several nonpathogenic Neisseria species, such as Neisseria cinerea and Neisseria polysaccharea (1, 4–10). This is most likely due to the high degree of genetic relatedness between N. meningitidis and the commensal Neisseria species. Therefore, the use of complementary laboratory techniques, including PCR, 16S rRNA sequencing, and traditional biochemical tests, is typically recommended for identification of N. meningitidis (3, 5, 7, 8, 11–13).
BC Children’s Hospital (BCCH) microbiology laboratory performs PCR directly on EDTA blood and cerebrospinal fluid (CSF) samples as well as using it as a confirmatory tool for clinical isolates identified as N. meningitidis by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany). Previously, BCCH’s PCR targeted ctrA, a capsule transport gene that was thought to be conserved in all invasive strains of N. meningitidis due to the role of the capsule in circumventing complement-mediated killing (14). However, the sensitivity of ctrA in identification of all invasive N. meningitidis isolates has been called into question. Several cases of invasive and sometimes fatal disease caused by capsule-null (cnl) strains lacking ctrA have been described, both in immunocompromised patients and in patients without an identifiable immune deficiency (15–18). Two confirmed cnl isolates from invasive disease demonstrated resistance to complement-mediated killing in vitro (15, 16). There is also evidence of capsule-independent virulence determinants that may be transferred horizontally to acapsular strains (15, 16, 19). Furthermore, N. meningitidis has a high degree of genomic diversity fueled by natural competence, frequent horizontal gene transfer, and high rates of rearrangement, which poses a risk to the use of any single gene for identification (20–22). Molecular assays targeting ctrA have also yielded false-negative results for invasive isolates that were subsequently confirmed to contain a ctrA allele that had low identity with published sequences (20, 23).
We have recently modified our protocol for the molecular detection of N. meningitidis as a result of a false negative bronchoalveolar lavage (BAL) isolate. We supplemented ctrA-targeted PCR with three noncapsular gene targets, including the superoxide dismutase sodC (19, 24), the phenol metabolism gene metA (25), and the sulfite exporter tauE (25). These PCR targets were validated in direct comparison to ctrA against 32 molecularly and serologically characterized clinical N. meningitidis isolates from different specimen types as well as previouly PCR-positive samples (Table 1). The panel represented a genetically diverse sampling of N. meningitidis strains, including serogroupable, nonserogroupable, commonly invasive, uncommonly invasive, and cnl mutant strains. Of these, PCR targeting ctrA detected only 23 out of the 32 samples tested, while PCR targeting metA, sodC, and tauE successfully detected the 9 ctrA-negative samples. PCR for tauE and sodC each failed to detect 2 samples, whereas metA identified all 32 samples. Further validation of PCR targeted against metA, sodC, and tauE showed 100% specificity when tested against a range of isolates and samples (Table 2), including samples positive for Haemophilus influenzae DNA, which were tested because the sodC gene in N. meningitidis is believed to have been acquired via horizontal transfer from H. influenzae (26).
TABLE 1.
Cycle threshold values of Neisseria meningitidis PCR panel tested for different N. meningitidis gene targets
| Sample | Specimen typea | Serogroupb | Serotypeb |
CT valuesc |
|||
|---|---|---|---|---|---|---|---|
| ctrA | tauE | Meta | sodC | ||||
| 1 | Throat | NE | 15 | Negative | 28.34 | 29.39 | 32.7 |
| 2 | Blood | NE | 15 | Negative | 29.06 | 29.75 | 30.99 |
| 3 | Sputum | NE | NT | Negative | 28.22 | 28.92 | 29.88 |
| 4 | Sputum | NE | 4 | Negative | 28.47 | 29.48 | 30.13 |
| 5 | Sputum | NE | NT | Negative | 28.7 | 29.09 | 30.15 |
| 6 | Eye | NE | 19 | Negative | 29.6 | 30.16 | 31.12 |
| 7 | BAL | NE | 4 | Negative | 28.88 | 29.66 | 30.36 |
| 8 | Eye | Z | NT | 29.42 | 29.4 | 29.99 | 31.03 |
| 9 | Eye | Z | 4 | 29.41 | 29.07 | 29.73 | 30.5 |
| 10 | Sputum | AA | NT | 27.93 | 28.15 | 28.9 | Negative |
| 11 | Eye | AA | 1,19 | 29.55 | 29.02 | 30.07 | 30.58 |
| 12 | Sputum | AA | 19 | 28.36 | 28.1 | 28.77 | 29.69 |
| 13 | Eye | AA | 15 | Negative | 28.98 | 29.6 | 30.49 |
| 14 | Eye | AA | 2a | 29.22 | 28.95 | 29.73 | 30.61 |
| 15 | Eye | E | NT | 28.78 | 28.38 | 29.13 | 29.91 |
| 16 | Sputum | E | NT | 29.13 | Negative | 29.56 | 30.54 |
| 17 | Eye | E | 19 | 29.84 | 29.49 | 30.38 | 31.46 |
| 18 | Sputum | E | NT | 29.17 | 28.78 | 29.67 | 29.96 |
| 19 | Eye | E | 1,19 | 28.67 | 28.07 | 28.94 | 29.92 |
| 20 | Wound | NG | NT | 28.65 | 28.18 | 28.81 | 28.87 |
| 21 | Eye | NG | NT | 29.61 | 28.88 | 29.87 | 29.92 |
| 22 | Sputum | NG | 14,19 | 29.38 | 28.74 | 29.6 | 30.25 |
| 23 | Urethra | NG | NT | 29.12 | 28.72 | 29.44 | 30.08 |
| 24 | Eye | NG | 19 | 29.18 | 28.69 | 29.52 | 30.23 |
| 25 | BAL | NE | 4 | Negative | 29.82 | 29.88 | 31.75 |
| 26 | Blood (direct) | W | ND | 31.67 | 31.6 | 29.55 | 30.57 |
| 27 | Blood (direct) | NG | NT | 38.92 | 38.17 | 39.02 | 39.62 |
| 28 | CSF (direct) | B | ND | 29.27 | 28.95 | 32.09 | 33.17 |
| 29 | CSF (direct) | NG | NT | 38.39 | Negative | 39.23 | Negative |
| 30 | CSF (direct) | Y | ND | 28.91 | 28.98 | 28.98 | 29.97 |
| 31 | CSF (direct) | B | ND | 27.48 | 28.67 | 28.07 | 29.91 |
| 32 | CSF (direct) | B | ND | 22.83 | 22.71 | 23.46 | 23.89 |
These are clinical isolates unless otherwise specified (i.e., direct samples).
bAA, autoagglutinable; NE, nonencapsulated; NG, nongroupable by bacterial agglutination; NT, nonserotypeable; ND, not done.
CT, cycle threshold.
TABLE 2.
Specificity check using previously characterized samples positive for Haemophilus influenzae, negative for Neisseria meningitidis using ctrA-targeted PCR and cultured isolatesa
| Source | Characteristic |
|---|---|
| CSF | Haemophilus influenzae DNA detected by PCR |
| CSF | Haemophilus influenzae DNA detected by PCR |
| CSF | Haemophilus influenzae DNA detected by PCR |
| CSF | Haemophilus influenzae DNA detected by PCR |
| CSF | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| CSF | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| CSF | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| CSF | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| Blood | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| Blood | Neisseria meningitidis DNA not detected using ctrA-targeted PCR |
| Isolate | Neisseria lactamica ATCC 23970 |
| Isolate | Neisseria sicca ATCC 29193 |
| Isolate | Neisseria subflava ATCC 14799 |
| Isolate | Neisseria gonorrhoeae ATCC 43069 |
| Isolate | Moraxella catarrhalis |
| Isolate | Moraxella catarrhalis |
| Isolate | Moraxella osloensis |
| Isolate | Eikenella corrodens |
| Isolate | Eikenella corrodens |
| Isolate | Eikenella corrodens |
| Isolate | Eikenella corrodens |
| Isolate | Haemophilus influenzae |
| Isolate | Haemophilus parainfluenzae |
| Isolate | Kingella kingae ATCC 23330 |
| Isolate | Achromobacter xylosoxidans |
| Isolate | Escherichia coli |
| Isolate | Serratia marcescens |
| Isolate | Pseudomonas aeruginosa |
| Isolate | Gardnerella vaginalis |
| Isolate | Staphylococcus aureus |
| Isolate | Staphylococcus epidermidis |
| Isolate | Streptococcus agalactiae |
| Isolate | Streptococcus mitis |
| Isolate | Streptococcus pneumoniae ATCC 49619 |
| Isolate | Streptococcus pyogenes |
| Isolate | Streptococcus pneumoniae |
| Isolate | Enterococcus faecalis |
| Isolate | Clostridium perfringens |
| Isolate | Cutibacterium acnes |
| Isolate | Fusobacterium nucleatum |
| Isolate | Bacteroides fragilis |
| Isolate | Candida albicans |
| Isolate | Candida parapsilosis |
| Isolate | Cryptococcus neoformans |
All were negative for tauE, metA, sodC, and ctrA genes.
Neisseria meningitidis is an important human pathogen for which false negative results can have devastating clinical and public health consequences. Laboratories should be aware that some commercial assays may target encapsulated strains only, which are now understood not to represent all strains capable of causing severe and invasive disease. As a result of our findings, our laboratory has adopted the noncapsular genes metA and sodC as complementary targets to ctrA in order to improve detection of N. meningitidis via multiplex PCR. Our results highlight the importance of using multiple complementary gene targets, including noncapsular genes, for the molecular identification of Neisseria meningitidis from clinical samples.
Contributor Information
Ghada N. Al-Rawahi, Email: galrawahi@cw.bc.ca.
Patricia J. Simner, Johns Hopkins
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