Keywords
diagnóstico molecular
reação em cadeia da polimerase em tempo real
vigilância epidemiológica
laboratório de saúde pública
How to Cite
Abstract
This paper describes the experience of the Instituto Adolfo Lutz (IAL) in implementing in-house real-time PCR (qPCR) assays used in the diagnosis of bacterial meningitis (BM) caused by Neisseria meningitidis (Nm), Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hi). Strategies for improving the originally proposed qPCR assays are presented, including the evaluation of different genetic targets and incorporation of internal quality control by human constitutive gene detection (RNase P) in biological samples processed in routine. It describes the qPCR assays established to identify the main Nm genogroups and the six Hi genotypes, which generate information that can contribute to evaluating the effectiveness of conjugate vaccines present in the National Immunization Program. It shows the transfer of qPCR assays to other Public Health Laboratories in the country and the contribution of using these assays in BM laboratory surveillance. Furthermore, it presents other IAL contributions to improve BM laboratory diagnosis, such as the production of genetic controls that are distributed to Brazilian public laboratories, the evaluation of the use of filter paper cards for transporting clinical samples between local and reference laboratories, and the evaluation of commercial kits used in the BM diagnosis. Finally, it mentions the final considerations of the work, with reflections on the future of using qPCR assays in the laboratory diagnosis of BM.
References
1. DATASUS. Tecnologia da informação a serviço do SUS. Meningite – casos confirmados notificados no sistema de informação de agravos de notificação – Brasil. 2024 [acesso em 20 de setembro 2024]. Disponível em:http://tabnet.datasus.gov.br/cgi/deftohtm.exe?sinannet/cnv/meninbr.def.
2. Organização Pan-americana de Saúde. OMS e parceiros pedem ação urgente contra meningite. 2021 [acesso em 10 de setembro 2024]. Disponível em:https://www.paho.org/pt/noticias/28-9-2021-oms-e-parceiros-pedem-acao-urgente-contra-meningite.
3. Centro de Vigilância Epidemiológica Prof. Alexandre Vranjac. Meningites. Dados estatísticos. 2024 [acesso em 20 de setembro 2024]. Disponível em: https://www.saude.sp.gov.br/cve-centro-de-vigilancia-epidemiologica-prof.-alexandre-vranjac/areas-de-vigilancia/doencas-de-transmissao-respiratoria/agravos/meningites.
4. World Health Organization. WHO Manual. Laboratory Methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. 2 ed. Genebra: WHO Press; 2011. Disponível em: http://whqlibdoc.who.int/hq/1999/WHO_CDS_CSR_EDC_99.7.pdf.
5. Salgado MM, Gonçalves MG, Fukasawa LO, Higa FT, Paulino JT, Sacchi CT. Evolution of bacterial meningitis diagnosis in Sao Paulo State-Brazil and future challenges. Arq Neuropsiquiatr. 2013;71(9B):672-6. doi: 10.1590/0004-282X20130148.
6. Brouwer MC, Thwaites GE, Tunkel AR, van de Beek D. Dilemmas in the diagnosis of acute community-acquired bacterial meningitis. Lancet. 2012;380(9854):1684-92. doi: 10.1016/S0140-6736(12)61185-4.
7. Diallo K, Feteh VF, Ibe L, Antonio M, Caugant DA, du Plessis M, et al. Molecular diagnostic assays for the detection of common bacterial meningitis pathogens: A narrative review. EBioMedicine. 2021;65:103274. doi: 10.1016/j.ebiom.2021.103274
8. 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(4):1553-8. doi: 10.1128/JCM.39.4.1553-1558.2001.
9. 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(6):e20675. doi: 10.1371/journal.pone.0020675.
10. Instituto Adolfo Lutz, Seção de Imunologia. Introdução da PCR convencional e em tempo real para o diagnóstico laboratorial das meningites bacterianas no Instituto Adolfo Lutz. BEPA. 2007;4(40):24-7.
11. Fukasawa LO, Gonçalves MG, Salgado MM, Araújo TP, Custódio AV, Lee H. Harrison, et al. Utilização da PCR em tempo real para o diagnóstico laboratorial rápido das meningites bacterianas. In: Anais do 25° Congresso Brasileiro de Microbiologia; 8-12 nov 2009; Porto de Galinhas, PE. Sociedade Brasileira de Microbiologia; 2009.
12. Thomas JD, Hatcher CP, Satterfield DA, Theodore MJ, Bach MC, Kristin B Linscott KB, et al. sodC-based real-time PCR for detection of Neisseria meningitidis. PLoS One. 2011;6(5):e19361. doi: 10.1371/journal.pone.0019361.
13. Higa FT, Fukasawa LO, Gonçalves MG, Salgado MM, Lemos APS, Harrison LH, et al. Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids. Mem Inst Oswaldo Cruz 2013;108(2):246-7. doi: 10.1590/0074-0276108022013020.
14. Weckx LY, Puccini RF, Machado A, Gonçalves MG, Tuboi S, de Barros E, et al. A cross-sectional study assessing the pharyngeal carriage of Neisseria meningitidis in subjects aged 1-24 years in the city of Embu das Artes, São Paulo, Brazil. Braz J Infect Dis. 2017;21(6):587-595. doi: 10.1016/j.bjid.2017.06.005.
15. Moraes JC, Kemp B, de Lemos AP, Outeiro Gorla MC, Lemes Marques EG, Ferreira Mdo C, et al. Prevalence, Risk Factors and Molecular Characteristics of Meningococcal Carriage Among Brazilian Adolescents. Pediatr Infect Dis J. 2015;34(11):1197-202. doi: 10.1097/INF.0000000000000853.
16. Sáfadi MA, Carvalhanas TR, Paula de Lemos A, Gorla MC, Salgado M, Fukasawa LO, et al. Carriage rate and effects of vaccination after outbreaks of serogroup C meningococcal disease, Brazil, 2010. Emerg Infect Dis. 2014;20(5):806-11. doi: 10.3201/eid2005.130948.
17. Almeida SCG, Fukasawa LO, Brandão AP, Salgado MM, Leite D, Brandileone MCC, et al. Importância dos genes ply e lytA na detecção molecular de Streptococcus pneumoniae por PCR em tempo real. In: Anais do I Simpósio Internacional de Microbiologia Clínica; 16-19 set 2008; Gramado, RS. Sociedade Brasileira de Microbiologia; 2008.
18. Sam IC, Smith M. Failure to detect capsule gene bexA in Haemophilus influenzae types e and f by real-time PCR due to sequence variation within probe binding sites. J Med Microbiol. 2005;54(Pt5):453-455. doi: 10.1099/jmm.0.45836-0.
19. Zanella RC, Bokerman S, Andrade ALSS, Flannery B, Brandileone MCC. Changes in serotype distribution of Haemophilus influenzae meningitis isolates identified through laboratory-based surveillance following routine childhood vaccination against H. influenzae type b in Brazil. Vaccine. 2011; 29(48):8937-42 doi: 10.1016/j.vaccine.2011.09.053.
20. Wang X, Mair R, Hatcher C, Theodore MJ, Edmond K, Wu HM, et al. Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae. Int J Med Microbiol. 2011;301(4):303-9. doi: 10.1016/j.ijmm.2010.11.004.
21. Salgado MM, Higa FT, Gonçalves MG, Fukasawa LO, Liphaus BL, Oliveira PL, et al. Nova versão do ensaio de PCR em tempo real para o diagnóstico laboratorial e vigilância epidemiológica das meningites bacterianas. BEPA 2012;9(103):16-20.
22. Gonçalves MG, Higa FH, Fukasawa LO, Barros LDA, Salgado MM. Evolução na vigilância laboratorial do Haemophilus influenzae nas meningites e pneumonias bacterianas, por PCR em tempo real, no Estado de São Paulo (2010-2019). BEPA 2022;19:1-16. doi: https://doi.org/10.57148/bepa.2022.v.19.37293.
23. 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. Emerg Infect Dis. 2004;10(2):311-6. doi: 10.3201/eid1002.030759.
24. Wang X, Theodore MJ, Mair R, Trujillo-Lopez E, du Plessis M, Wolter N, et al. Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens. J Clin Microbiol 2012;50(3):702-8. doi: 10.1128/JCM.06087-11.
25. Maaroufi Y, de Bruyne JM, Heymans C, Crokaert F. Real-time PCR for determining capsular serotypes of Haemophilus influenzae. J. Clin. Microbiol 2007;45(7):2305-8. doi: 10.1128/JCM.00102-07.
26. Marasini D, Whaley MJ, Jenkins LT, Hu F, Jiang W, Topaz N, et al. Direct real-time PCR for the detection and serotyping of Haemophilus influenzae without DNA extraction. J Clin Microbiol. 2022;60(4):e0211121. doi: 10.1128/jcm.02111-21.
27. Pimenta FC, Roundtree A, Soysal A, Bakir M, du Plessis M, Wolter N, et al. Sequential triplex real-time PCR assay for detecting 21 pneumococcal capsular serotypes that account for a high global disease burden. J Clin Microbiol 2013;51(2):647-52. doi: 10.1128/JCM.02927-12.
28. Velusamy S, Tran T, Mongkolrattanothai T, Walker H, McGee L, Beall B. Expanded sequential quadriplex real-time polymerase chain reaction (PCR) for identifying pneumococcal serotypes, penicillin susceptibility, and resistance markers. Diagn Microbiol Infect Dis. 2020;97(2):115037. doi: 10.1016/j.diagmicrobio.2020.115037.
29. Zanella RC, Brandileone MCC, Almeida SCG, Lemos APS, Sacchi CT, Gonçalves CR, et al. Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in a Brazilian elderly cohort. PLoS One. 2019;14(8):e0221525. doi: 10.1371/journal.pone.0221525.
30. Brandileone MC, Zanella RC, Almeida SCG, Cassiolato AP, Lemos APS, Salgado MM, et al. Long-term effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae in children in Brazil. Vaccine. 2019;37(36):5357-63. doi: 10.1016/j.vaccine.2019.07.043.
31. Liphaus BL, Okai MICG, Lemos APS, Gorla MC, Fernandes MR, Pacola MR, et al. Outbreak of Neisseria meningitidis C in a Brazilian oil refinery involving an adjacent community. Enferm Infecc Microbiol Clin 2013;31(2):88–92. doi: 10.1016/j.eimc.2012.05.009.
32. Iser BPM, Lima HCAV, Moraes C, Almeida RPA, Watanabe LT, Alves SLA, et al. Outbreak of Neisseria meningitidis C in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community Epidemiol Infect. 2012;140(5):906-15. doi: 10.1017/S0950268811001610.
33. Fernandes RMBP, Doro CM, Reis R, Silva APM, Souza DF, Barbosa HA, et al. Doença meningocócica: investigação de surto comunitário no Distrito Administrativo do Ipiranga, município de São Paulo, julho de 2007. BEPA 2007;4(44): 10-7.
34. Fukasawa LO, Liphaus BL, Gonçalves MG, Higa FT, Camargo CH, Carvalhanas TRMP, et al. Invasive Meningococcal X Disease during the COVID-19 Pandemic, Brazil. Emerg Infect Dis. 2022;28(9):1931-2. doi: 10.3201/eid2809.220531.
35. Fukasawa LO, Sato NS, Sacchi CT. Construction of plasmids for use in real time PCR assays for the multiplex detection of bacterial meningitis pathogens. In: Anais do 5º Simpósio Internacional de Microbiologia Clínica; 13-15 mai 2016; São Pedro, SP. Sociedade Brasileira de Microbiologia; 2016.
36. Fukasawa LO, Sato NS, Sacchi CT. Construção de controles genéticos para melhoria da qualidade dos ensaios de PCR em tempo real para o diagnóstico de meningite bacteriana. Revista O Biológico. 2016;78(2).
37. Fukasawa LO, Gonçalves MG, Higa FT, Castilho EA, Ibarz-Pavón AB, Sacchi CT. Use of cerebrospinal fluid and serum samples impregnated on FTATM Elute filter paper for the diagnosis of infections caused by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. PLoS One. 2017;12(2):e0172794. doi: 10.1371/journal.pone.0172794.
38. Gonçalves MG, Higa FT, Fukasawa LO, Carvalho GA, Milagres BS, Salgado MM. Avaliação de kits comerciais baseados em PCR multiplex em tempo real para diagnóstico de meningite bacteriana. BEPA. 2023;20:e39209. doi: https://doi.org/10.57148/bepa.2023.v.20.39209.
39. Long JR, Mitchell K, Edwards J, Wroblewski D, Luke E, Dickinson M, et al. Laboratory diagnosis of bacterial meningitis by direct detection, serotyping and Next Generation Sequencing: How 10 years of testing in New York State has evolved to improve laboratory diagnosis and public health. Mol Cell Probes. 2022;61:101786. doi: 10.1016/j.mcp.2021.101786.
40. Côrtes LGF, Maldonado MM, Koga PCM, Santiago KAS, Fernandes GBP, Maluf MM, et al. Evaluation of pathogen from the FilmArray meningitis/encephalitis panel and recommendations on atypical findings Arq Neuropsiquiatr. 2024;82(1):1-8. doi: 10.1055/s-0044-1779035.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2024 Lucila Okuyama Fukasawa, Maria Gisele Gonçalves, Fábio Takenori Higa, Maristela Marques Salgado, Cláudio Tavares Sacchi