Avaliação de resultados discrepantes obtidos na execução de PCR em tempo real em amostras de pacientes com suspeita clínica de meningite bacteriana

Maristela Marques Salgado; Maria Gisele Gonçalves; Fábio Takenori Higa; Lucila Okuyama Fukasawa; Priscilla Lima de Oliveira; Carla Naufal da Silva; Cláudio tavares Sacchi

doi:10.18241/0073-98552013721558

Vol. 72 No. 2 (2013), BRIEF COMMUNICATION

Vol. 72 No. 2 (2013)

Inconsistent results in real-time PCR in analyzing samples from patients with suspected bacterial meningitis

BRIEF COMMUNICATION

https://doi.org/10.18241/0073-98552013721558

Published 2012-02-22

Maristela Marques Salgado⁺⁻
Maria Gisele Gonçalves⁺⁻
Fábio Takenori Higa⁺⁻
Lucila Okuyama Fukasawa⁺⁻
Priscilla Lima de Oliveira⁺⁻
Carla Naufal da Silva⁺⁻
Cláudio tavares Sacchi⁺⁻

Maristela Marques Salgado

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

Maria Gisele Gonçalves

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

https://orcid.org/0000-0002-8966-5927

Fábio Takenori Higa

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

Lucila Okuyama Fukasawa

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

Priscilla Lima de Oliveira

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

Carla Naufal da Silva

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

Cláudio tavares Sacchi

Instituto Adolfo Lutz, Centro de Imunologia, São Paulo, SP

PDF (Português (Brasil))

Keywords

real-time polymerase chain reaction
bacterial meningitis
laboratory diagnosis
N. meningitidis
S. pneumoniae
H. influenzae

How to Cite

1.

Salgado MM, Gonçalves MG, Higa FT, Fukasawa LO, Oliveira PL de, Silva CN da, Sacchi C tavares. Inconsistent results in real-time PCR in analyzing samples from patients with suspected bacterial meningitis. Rev Inst Adolfo Lutz [Internet]. 2012 Feb. 22 [cited 2024 Jul. 22];72(2):161-4. Available from: https://periodicos.saude.sp.gov.br/RIAL/article/view/32912

Abstract

The basic principle for obtaining a reliable result is the consistency found among the replicas and its reproducibility. In a diagnostic routine by using real-time PCR (RT-PCR), when hundreds samples are processed, and results with late Cts or replicas that differ by more than three units are inevitable. Of 3,000 samples processed in 2010, in duplicate, in the diagnostic routine of bacterial meningitis by RT-PCR for detecting N. meningitidis, S. pneumoniae and H. influenzae, 157 (5.2 %) samples showed inconsistency among replicas (difference between Cts higher than three units) and/or high Cts values; and the samples were retested. This study assessed these results, the benefits of its repetitions and probable reasons for the occurrence of these discrepant results. Among the retested samples, 18 (11 %) only showed positive results. Human errors inherent to pipetting, use of non-calibrated pipettes, low concentration of target DNA in the analyzed samples, probe degradation or even random contamination are factors which contribute to induce the discrepant results. Performing RT-PCR assay with samples in duplicate and retesting the samples showing discordant results constitute a device for efficiently evaluating and defining these results.

https://doi.org/10.18241/0073-98552013721558

PDF (Português (Brasil))

References

1. Wu HM, Cordeiro SM, Harcourt BH, Carvalho MGS, Azevedo J, Oliveira TQ, et al. Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis. BMC Infect Dis. 2013;13:26-44.

2. Fukasawa LO, Salgado MM, Marques EGL, Fernandes RMBP, Kemp B, Carvalhanas TR. Validação da técnica de contraimunoeletroforese (CIE) para o diagnóstico laboratorial das meningites causadas por Neisseria meningitidis sorogrupos A, B, C e W135. BEPA. 2012;9(102):13-20.

3. Sacchi CT, Fukasawa LO, Gonçalves MG, Salgado MM, Shutt KA, Carvalhanas TR, et al. Incorporation of Real-Time PCR into Routine Public Health Surveillance of Culture Negative Bacterial Meningitis in São Paulo, Brazil. PLoS ONE. 2011;6:1-8.

4. Mothershed EA, Sacchi CT, Whitney AM, Barnett GA, Ajello GW, Schmink S, et al. Use of real-time PCR to resolve slide agglutination discrepancies in serogroup identification of Neisseria meningitidis. J Clin Microbiol. 2004;42:320-8.

5. Carvalho MGS, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW, et al. Evaluation and improvement of real-time PCR detection assays to lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol. 2007;45:2460-6.

6. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol. 2001;39:1553-8.

7. Emery SL, Erdman DD, Bowen MD, Newton BR, Winchell JM, Meyer RF, et al. Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus. Emerging Infect Dis. 2004;10:311-6.

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Inconsistent results in real-time PCR in analyzing samples from patients with suspected bacterial meningitis

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