Validation of multiplex PCR for the diagnosis of acute bacterial meningitis in culture negative cerebrospinal fluid

Almeida, Sérgio Monteiro de; Costa, Libera Maria Dalla; Siebra, Christian; Arend, Lavinia Nery Villa Stangler; Nogueira, Keite da Silva

doi:10.1590/0004-282x20190028

Cited by 11 publications

(9 citation statements)

References 32 publications

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“… 5 FilmArray was shown to be efficatious in detecting bacterial etiology in meningitis cases in which CSF culture was negative. 6 , 7 A previous report showed rapid detection of Listeria monocytogenes in a patient in which CSF Gran stain and culture were negative, as occurred in a case in our series. 8 A large study showed high sensitivity and specificity levels.…”

supporting

confidence: 67%

FilmArray Meningitis/Encephalitis (ME) panel in the diagnosis of bacterial meningitis

Domingues¹,

Santos²,

Leite³

et al. 2019

The Brazilian Journal of Infectious Diseases

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supporting

confidence: 67%

FilmArray Meningitis/Encephalitis (ME) panel in the diagnosis of bacterial meningitis

Domingues¹,

Santos²,

Leite³

et al. 2019

The Brazilian Journal of Infectious Diseases

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“…This difference in results was statistically significant (P < 0.05). complement to classical bacteriology techniques [29]. PCR showed in this study a significantly higher detection rate of bacterial meningitis when compared to culture, the confirmation rate was 33% by culture and 70% by RT-PCR.…”

Section: Discussionsupporting

confidence: 54%

Simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae by quantitative PCR from CSF samples with negative culture in Morocco

Ikken

Benaouda²,

Yaich³

et al. 2021

AMicr

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Bacteriological cultures from cerebrospinal fluids (CSF) have less sensitivity and specificity compared to quantitative PCR (RT-PCR), and multiple facts still conduct to the increase of negative culture. The aims of this study are to determine the molecular epidemiology and the simultaneous detection of bacterial meningitis in Morocco by using RT-PCR and compared this molecular approach with culture method to improve the etiological diagnosis of meningitis. The CSFs were collected over one-year period in 2018 in different hospitals covering all regions of the Kingdom of Morocco, from patients with suspected meningitis. The results showed the confirmation rate per culture recorded a rate of 33% and the RT-PCR of 70%. Molecular epidemiology is predominant of Neisseria meningitidis followed by Streptococcus pneumoniae and a dramatic reduction in meningitis due to Haemophilus influenzae following the introduction of conjugate vaccine in 2007. Also, the epidemiological profile shows a sex ratio M/F of 1.4 and a median age of 2 years. The national distribution showed a predominant of meningococcal disease followed by pneumococcal disease, especially a dominance of N. meningitidis over S. pneumoniae in two regions and a slight predominance of S. pneumoniae in the other two regions over N. meningitidis. Our research shows that culture in our country has less sensitivity and specificity than RT-PCR in diagnosis of bacterial meningitis and that molecular biology technique at bacteriology laboratories is desirable for diagnosis, early management of meningitis cases and in the context of the surveillance of meningitis in Morocco in parallel with culture.

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“…qPCR has high sensitivity and specificity, and produces faster results because it does not require the previous growth of the microorganism. Multiplex qPCR of the cerebrospinal fluid (CSF) proved to be a valuable method for improving the rapidity and accuracy of a diagnosis of bacterial meningitis, even in cases with CSF culture-negative results [ 4 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

“…Real-time PCR is highly accurate in diagnosing meningitis caused by S . pneumoniae [ 4 , 5 , 7 ] and it also can be applied as a tool to detect antimicrobial resistance genes faster than the identification of phenotype by AST, especially in culture-negative samples.…”

Section: Introductionmentioning

confidence: 99%

Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

Souza

Cergole-Novella²,

Molinari³

et al. 2022

PLoS ONE

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Meningitis caused by Streptococcus pneumoniae is still a disease of great impact on Public health, which requires immediate diagnosis and treatment. However, the culture of clinical specimens is often negative and antibiotic susceptibility testing (AST) must be performed with isolated strains. Multiplex real-time polymerase chain reaction (qPCR) has high sensitivity and specificity, produces faster results to identify the pathogen, and it can also be an important tool to identify resistance antibiotic genes earlier than AST, especially in the absence of an isolated strain. This study developed a multiplex qPCR assay, using SYBR Green as a nonspecific dye, to detect antibiotic resistance genes to predict pneumococcal susceptibility/resistance in cerebrospinal fluid (CSF) samples from meningitis patients. From 2017 to 2020, CSF samples were cultured and analyzed by qPCR to detect the main three bacteria causing meningitis. Isolated and reference strains were applied in SYBR Green qPCR multiplex to detect pbp2b, ermB, and mef genes, and the results were compared with the AST. Pneumococcal-positive CSF samples (lytA-positive gene) without isolated strains were also tested to evaluate the antimicrobial susceptibility profile in the region from 2014 to 2020. From the received 873 CSF samples; 263 were cultivated, 149 were lytA-positive in the qPCR, and 25 produced viable isolated pneumococci strains, which were evaluated by AST. Melting temperature for each gene and the acceptance criteria were determined (pbp2b: 78.24–79.86; ermB: 80.88–82.56; mef: 74.85–76.34 ºC). A total of 48/51 strains presented a genetic profile in agreement with the AST results. Resistant strains to erythromycin and clindamycin were ermB-positive, and two were also mef-positive, indicating both resistance mechanisms were present. In the retrospective study of the genetic profile of resistance, 82 lytA-positive CSF samples plus 4 strains were applied in the SYBR Green qPCR multiplex: 51% of samples presented the wild genotype (pbp2b positive and ermB/mef negative); 15% were negative for all the three evaluated, indicating pneumococci resistant to penicillin; and 17% represented the multidrug-resistant pneumococci (pbp2b negative and ermB positive or pbp2b negative and ermB and mef positive). Therefore, SYBR Green qPCR multiplex proved to be a reliable tool to identify resistance genes in S. pneumoniae and would be less expensive than multiplex qPCR using specific probes. This could be easily introduced into the routine of diagnostic laboratories and provide a strong presumption of pneumococcal resistance, especially in the absence of isolated strains.

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Validation of multiplex PCR for the diagnosis of acute bacterial meningitis in culture negative cerebrospinal fluid

Cited by 11 publications

References 32 publications

FilmArray Meningitis/Encephalitis (ME) panel in the diagnosis of bacterial meningitis

FilmArray Meningitis/Encephalitis (ME) panel in the diagnosis of bacterial meningitis

Simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae by quantitative PCR from CSF samples with negative culture in Morocco

Multiplex real-time PCR using SYBR Green: Unspecific intercalating dye to detect antimicrobial resistance genes of Streptococcus pneumoniae in cerebrospinal fluid

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