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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2010, p. 4590–4591 Copyright 2010, American Society for Microbiology. All Rights Reserved.
False-Negative PCR Result Due to Gene Polymorphism: the Example ´atrice Ninet,2 Jacques Bille,1 and Gilbert Greub1 Institute of Microbiology, University Hospital of Lausanne, Lausanne, Switzerland,1 and Bacteriology Laboratory, National Center of Meningococci, University Hospital of Geneva, Geneva, Switzerland2 Received 31 August 2010/Accepted 11 October 2010 Early treatment of meningococcal meningitis is mandatory but may negate the cerebrospinal fluid culture.
Etiological diagnosis then mainly relies on PCR. Here, we report a case of false-negative results for real-time
PCR for a Neisseria meningitidis
serogroup B isolate with a polymorphism in the ctrA gene.
Bacterial meningitis is life threatening, and rapid treatment CSF puncture. Gram-negative diplococci were seen on Gram is mandatory. A number of culture-negative meningococcal staining of CSF, but the culture remained sterile. The real-time disease cases have been observed in many countries due to PCR was done on CSF and was surprisingly negative. Then, a increasing use of preadmission antibiotics. Therefore, detec- broad-spectrum 16S RNA PCR was performed (8), and N. tion of meningococcal DNA by PCR is widely used for patients meningitidis DNA was detected in the CSF. Meanwhile, the with suspected meningococcal meningitis and negative cere- blood cultures became positive for N. meningitidis serogroup B brospinal fluid (CSF) cultures (2). In this context, we read with susceptible to beta-lactams. The clinical progression was favor- great interest the report by Cavrini et al. (3) on multiple nu- able, and the child was sent home after 5 days of hospitaliza- cleotide substitutions in two isolates of Neisseria meningitidis serogroup C causing false-negative detection, with a real-time The blood isolate was identified as N. meningitidis by an API We encountered a similar problem with a clinical isolate of grouped using the Pastorex meningitis antiserum panel (Bio- N. meningitidis serogroup B not detected by our real-time PCR Rad, Marnes-la-Coquette, France). It was more precisely targeting the ctrA gene. For 5 years, we have been using me- typed at the Swiss Reference Center of Meningococci. The ningococcal DNA detection by PCR for patients with sus- serogroup was defined with an agglutination test, the serotype pected meningococcal meningitis and negative CSF cultures,as PCR increases the number of confirmed cases (4, 6). We use and subtype were defined with a dot enzyme-linked immu- the primers and probe described by Corless et al. (4), also used nosorbent assay (dot-ELISA) method (7), and the multilocus by Cavrini et al. and by several other groups (6, 9, 10, 15, 16).
sequence type was defined by sequencing seven housekeeping This primer and probe combination was tested for its analytical genes (1). This strain was confirmed to belong to serogroup B, specificity on many N. meningitidis strains (9, 15). Before in- B:Ϫ:P1.2,5, and ST-269. However, when the DNA, extracted troducing it in our diagnostic laboratory, this real-time PCR from the blood culture isolate, was tested by the real-time ctrA was evaluated on a collection of 37 CSF samples (11 positive N. meningitidis PCR, the result was again negative without for N. meningitidis, 10 positive for Streptococcus pneumoniae, 3 positive for Listeria monocytogenes, 1 positive for Haemophilus Following these results, the complete ctrA gene was ampli- influenzae, 6 positive for other bacteria, and 6 negative for any fied with one primer located in the operon for capsule biosyn- bacteria) with specificity and sensitivity of 100%.
thesis (siaA), NM_SEQ1F (5Ј-TCTGGTACCTGTAATGCA Then, from 2005 to July 2010, a total of 419 CSF samples AAGAATTC-3Ј), and another primer in the ctrB gene, received with a diagnostic of meningitis were examined by PCR. During these 5 years, 5 samples were PCR and culture C-3Ј), in order to include the entire ctrA gene. On the agarose positive for N. meningitidis, whereas 17 were positive by PCR gel, a single band was observed with a much larger size (2,322 but negative by culture. Among the remaining 397 PCR-neg- bp) than the one obtained with positive-control N. meningitidis ative CSF samples, one specimen was documented as being isolates (1,485 bp). The segment was sequenced with the prim- falsely negative with the N. meningitidis ctrA PCR.
ers used for amplification as well as with the following internal The patient, a 2-year-old boy, presented to the hospital with primers: NM_NM-F (5Ј-GCTGCGGTAGGTGGTTCAA-3Ј) meningitis and petechiae. He was already being treated with amoxicillin per os. Blood cultures were drawn, and ceftriaxone Analysis of the obtained sequence revealed interesting fea- and dexamethasone were administered intravenously before tures. (i) The ctrA gene (bp 1005 to 2180) exhibited only 97%identity with all published available N. meningitidis ctrA gene * Corresponding author. Mailing address: Institute of Microbiology, sequences, except the 2 strains recently described by Cavrini et University Hospital of Lausanne (CHUV), Bugnon 48, 1011 Lausanne, al. (3); our strain also presented a strong polymorphism on the Switzerland. Phone: 41 79 556 1680. Fax: 41 21 314 4060. E-mail: reverse primer and a single nucleotide substitution on the ᰔ Published ahead of print on 20 October 2010.
probe, which precluded PCR amplification. (ii) Another fea- ture was an insertion segment, IS1301 (837 bp), in the sia/ctr 2. Brouwer, M. C., A. R. Tunkel, and D. van de Beek. 2010. Epidemiology,
diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin.
Microbiol. Rev. 23:467–492.
To assess the frequency of such polymorphism, we tested by 3. Cavrini, F., G. Liguori, A. Andreoli, and V. Sambri. 2010. Multiple nucleo-
our real-time PCR our collection of N. meningitidis clinical tide substitutions in the Neisseria meningitidis serogroup C ctrA gene cause
false-negative detection by real-time PCR. J. Clin. Microbiol. 48:3016–3018.
isolates since 2000 (n ϭ 38) and none exhibited this polymor- 4. Corless, C. E., M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, and E. B.
phism. Another attempt was to focus on the N. meningitidis Kaczmarski. 2001. Simultaneous detection of Neisseria meningitidis, Hae-
strains belonging to the ST-269 complex. These strains are very mophilus influenzae, and Streptococcus pneumoniae in suspected cases of
meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39:1553–
rare in Switzerland, less than one isolate being found per year, but they account for 6% of all isolates described in the EU- 5. El Aila, N. A., S. Emler, T. Kaijalainen, T. De Baere, B. Saerens, E. Alkan,
MenNet project (1). The four serogroup B, ST-269 isolates P. Deschaght, R. Verhelst, and M. Vaneechoutte. 2010. The development of
a 16S rRNA gene based PCR for the identification of Streptococcus pneu-
collected from 2004 to 2009 in Switzerland were tested by the moniae and comparison with four other species specific PCR assays. BMC ctrA real-time PCR, and the result was negative for 2 of them.
Infect. Dis. 10:104.
Those 2 strains belong to the same serotype and serosubtype, 6. Fernandez-Rodriguez, A., B. Alcala, and R. Alvarez-Lafuente. 2008. Real-
time polymerase chain reaction detection of Neisseria meningitidis in for- B:Ϫ:P1.2,5, as does the strain isolated from the index case malin-fixed tissues from sudden deaths. Diagn. Microbiol. Infect. Dis. 60:
reported above. Both strains exhibited 100% identity with the index ctrA sequence isolate as demonstrated by sequencing the Frasch, C. E., W. D. Zollinger, and J. T. Poolman. 1985. Serotype antigens
of Neisseria meningitidis and a proposed scheme for designation of sero-
polymorphic 3Ј-end region of the ctrA gene (567 bp).
types. Rev. Infect. Dis. 7:504–510.
To check whether we could have missed additional N. menin- 8. Goldenberger, D., A. Kunzli, P. Vogt, R. Zbinden, and M. Altwegg. 1997.
Molecular diagnosis of bacterial endocarditis by broad-range PCR amplifi- gitidis DNA-positive CSF samples with negative culture results, a cation and direct sequencing. J. Clin. Microbiol. 35:2733–2739.
newly designed PCR was applied to 93 CSF samples collected 9. Guiver, M., R. Borrow, J. Marsh, S. J. Gray, E. B. Kaczmarski, D. Howells,
from January 2009 to July 2010 with the same forward primer but P. Boseley, and A. J. Fox. 2000. Evaluation of the Applied Biosystems
automated Taqman polymerase chain reaction system for the detection of
a new reverse primer, NM_NM2-R (5Ј-ACCACGGCGCAACA meningococcal DNA. FEMS Immunol. Med. Microbiol. 28:173–179.
AAATA-3Ј), and a new probe, NM_NM2-P (5Ј-VIC-CATTGC 10. Hedberg, S. T., P. Olcen, H. Fredlund, and P. Molling. 2009. Real-time PCR
CACGTGTTAGCTGCACAT-BHQ-3Ј), corresponding to the detection of five prevalent bacteria causing acute meningitis. APMIS 117:
856–860.
polymorphism. None was positive, suggesting that this polymor- 11. Jefferies, J., L. Nieminen, L. A. Kirkham, C. Johnston, A. Smith, and T. J.
phism is relatively rare. Nevertheless, we now use both ctrA prim- Mitchell. 2007. Identification of a secreted cholesterol-dependent cytolysin
ers/probes (the one reported by Corless et al. [4] and the one with (mitilysin) from Streptococcus mitis. J. Bacteriol. 189:627–632.
12. Kugelberg, E., B. Gollan, C. Farrance, H. Bratcher, J. Lucidarme, A. B.
the NM_NM2-R as reverse primer and the NM_NM2-P probe) Ibarz-Pavon, M. C. Maiden, R. Borrow, and C. M. Tang. 2010. The influence
in order not to miss additional cases exhibiting such a polymor- of IS1301 in the capsule biosynthesis locus on meningococcal carriage and
disease. PLoS One 5:e9413.
13. Riffelmann, M., C. H. Wirsing von Konig, V. Caro, and N. Guiso. 2005.
In conclusion, isolates with polymorphism or closely related Nucleic acid amplification tests for diagnosis of Bordetella infections. J. Clin.
isolates may generate false-negative or false-positive PCR re- Microbiol. 43:4925–4929.
14. Ripa, T., and P. A. Nilsson. 2007. A Chlamydia trachomatis strain with a
sults, especially when a single gene is targeted, as demon- 377-bp deletion in the cryptic plasmid causing false-negative nucleic acid strated here as well as previously for Chlamydia trachomatis amplification tests. Sex. Transm. Dis. 34:255–256.
(14), Streptococcus pneumoniae (5, 11, 17), and Bordetella per- 15. Taha, M. K., J. M. Alonso, M. Cafferkey, D. A. Caugant, S. C. Clarke, M. A.
Diggle, A. Fox, M. Frosch, S. J. Gray, M. Guiver, S. Heuberger, J. Kal-
tussis (13). To target at least two different genes may overcome musova, K. Kesanopoulos, A. M. Klem, P. Kriz, J. Marsh, P. Molling, K.
this risk. Microbiologists should be aware of this problem and Murphy, P. Olcen, O. Sanou, G. Tzanakaki, and U. Vogel. 2005. Interlabo-
should alert the scientific community when such events occur.
ratory comparison of PCR-based identification and genogrouping of Neis-
seria meningitidis. J. Clin. Microbiol. 43:144–149.
Nucleotide sequence accession number. The sequence of the
16. Tzanakaki, G., M. Tsopanomichalou, K. Kesanopoulos, R. Matzourani, M.
complete ctrA gene has been submitted under accession num- Sioumala, A. Tabaki, and J. Kremastinou. 2005. Simultaneous single-tube
PCR assay for the detection of Neisseria meningitidis, Haemophilus influ-
enzae type b and Streptococcus pneumoniae. Clin. Microbiol. Infect. 11:386–
390.
´ Brouillet for excellent technical work.
17. Whatmore, A. M., A. Efstratiou, A. P. Pickerill, K. Broughton, G. Woodard,
D. Sturgeon, R. George, and C. G. Dowson. 2000. Genetic relationships
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