Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery

Perdigão Neto, Lauro Vieira; Medeiros, Micheli; Ferreira, Suzete Cleusa; Nishiya, Anna Shoko; de Assis, Denise Brandão; Boszczowski, Ícaro; Costa, Silvia Figueiredo; Levin, Anna S.

doi:10.6061/clinics/2021/e2284

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ORIGINAL ARTICLE • Clinics 76 • 2021 • https://doi.org/10.6061/clinics/2021/e2284 copy

Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery

Authorship SCIMAGO INSTITUTIONS RANKINGS

OBJECTIVES:

Bacterial and aseptic meningitis after neurosurgery can present similar clinical signs and symptoms. The aims of this study were to develop and test a molecular method to diagnose bacterial meningitis (BM) after neurosurgery.

METHODS:

A 16S ribosomal RNA gene PCR-based strategy was developed using artificially inoculated cerebrospinal fluid (CSF) followed by sequencing. The method was tested using CSF samples from 43 patients who had undergone neurosurgery and were suspected to suffer from meningitis, and from 8 patients without neurosurgery or meningitis. Patients were classified into five groups, confirmed BM, probable BM, possible BM, unlikely BM, and no meningitis.

RESULTS:

Among the samples from the 51 patients, 21 samples (41%) were culture-negative and PCR-positive. Of these, 3 (14%) were probable BM, 4 (19%) were possible BM, 13 (62%) were unlikely BM, and 1 (5%) was meningitis negative. Enterobacterales, non-fermenters (Pseudomonas aeruginosa and Acinetobacter baumannii), Staphylococcus haemolyticus, Granulicatella, Variovorax, and Enterococcus cecorum could be identified. In the group of patients with meningitis, a good agreement (3 of 4) was observed with the results of cultures, including the identification of species.

CONCLUSION:

Molecular methods may complement the diagnosis, guide treatment, and identify non-cultivable microorganisms. We suggest the association of methods for suspected cases of BM after neurosurgery, especially for instances in which the culture is negative.

Meningitis, Bacterial; Craniotomy; Cerebrospinal Fluid; Diagnosis; RNA, Ribosomal, 16S; Sequence Analysis

Patients treated by neurosurgery (n=43)
Male	n=23 (53%)
Age (years)	Mean: 52.5 (range:18-87)
Neurological conditions
CNS tumors	n=12 (28%)
Hydrocephalus	n=6 (14%)
Subarachnoid hemorrhage	n=6 (14%)
Head trauma	n=5 (11%)
Cerebellar/brain hematomas	n=4 (9%)
Stroke	n=2 (5%)
Neurocysticercosis	n=2 (5%)
Cist	n=2 (5%)
Vertebral instability	n=2 (5%)
Meningioma	n=1 (2%)
Lesion in fourth ventricle	n=1 (2%)
CSF cell count	Mean: 475 cells/mm³ (range: 0-4904)
CSF protein level	Mean: 163 mg/dL (range: 10-735)
CSF lactate concentration	Mean: 44 mg/dL (range: 11-162)
Glucose level	Mean: 60 mg/dL (range: 3-206)

Patients treated by neurosurgery (n=43)
Male	n=2 (25%)
Age (years)	Mean: 43 (range:39-47)

Definition Number of patients	Positive by PCR	Positive by culture	PCR amplicon sequencing
G1 Confirmed bacterial meningitis n=4	3	Klebsiella pneumoniae (n=2) Enterobacter cloacae (n=1) Staphylococcus haemolyticus (n=1)	K. pneumoniae (n=1) E. cloacae (n=1) S. haemolyticus (n=1)
G2 Probable bacterial meningitis n=5	3	-	Variovorax boronicumulans (n=2) Enterococcus cecorum (n=1)
G3 Possible bacterial meningitis n=10	4	-	Granulicatella adiacens (n=1) Pseudomonas aeruginosa (n=1) *Unidentified (n=2)
G4 Bacterial meningitis unlikely n=24	13	-	G. adiacens (n=1) Variovorax paradoxos (n=1) P. aeruginosa (n=1) Acinetobacter baumannii (n=1) Enterobacterales (n=3) *Unidentified (n=6)
G5 No meningitis n=8	1	-	*Unidentified (n=1)
Total n=51	24 (47%)

Author, year	Objective	Methods	Patients (n)	Main results and conclusions
Rath, 2014	To evaluate the clinical utility of RT-PCR of CSF in combination with IL-6 and lactate and compare them to CSF culture for the diagnosis of EVD-related ventriculitis/meningitis.	Leucocyte cell count, concentrations of glucose, protein, IL-6 and lactate, RT-PCR and culture of CSF of patients with possible ventriculitis/meningitis related to EVD were performed.	42	-Agreement of 92% of culture and PCR-based method. Discrepant results were mostly considered as contaminants. -PCR-based method is suitable for the identification of microoganisms in CSF of neurosurgery patients, especially if combined with biomarkers and lactate.
Deutch, 2007	To compare a RT-PCR diagnostic strategy with culture to evaluate additional effects on diagnosis and quantification of bacterial load in ventricular drainage-related bacterial meningitis.	CSF samples from patients with EVD or ventriculoperitoneal shunt during the course of bacterial meningitis were analyzed by r16S PCR and cultures.	86	-16 patients had any positive sample in culture or PCR. Four episodes of bacterial meningitis were diagnosed via PCR alone (predominantly caused by gram-negative), five episodes via culture alone, and seven episodes via both culture and PCR. -Culture supplemented with RT-PCR may increase the number of etiologically diagnosed bacterial meningitis episodes.
Banks, 2005	To report the analysis of CSF from ventriculoperitoneal shunts or EVD using both conventional cultures and PCR.	CSF samples from patients who underwent either shunt tap or routine surveillance cultures of their ventriculostomy, with clinical suspicion of infection, were tested using standard culture techniques and r16S PCR.	28	-21% of the samples were positive using both the culture and PCR; 30% were negative in PCR and culture; 49% of the samples were culture-negative and PCR-positive. -Cutibacterium acnes (fomerly Propionibacterium acnes) and S. aureus were the most frequent agents. -The data suggest that PCR is a highly sensitive, rapid, and promising modality for the detection and treatment of CSF shunt ventriculostomy infection.
Druel, 1996	To evaluate the presence of bacteria in samples from patients suffering from ‘aseptic' meningitis following craniotomy.	CSF from patients suffering from post-craniotomy meningitis and negative control patients were submitted to conventional culture and to PCR using bacterial r16S universal primers, followed by phylogenetic analysis.	24	-CSF from patients with either culture-positive or culture-negative meningitis yielded positive amplifications, whereas no amplification was obtained with CSF from control patients. -Six PCR products were cloned and sequenced: Pseudomonas (n=3), Escherichia (n=2), and Rhodococcus (n=1). -Many cases of culture-negative (aseptic) meningitis are probably bacterial meningitis and justify antibiotic treatment.

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[1] *Corresponding author. E-mail: lauro_perdigao@hotmail.com