May bacterial meningitis pave the way for a toxoplasmosis flare-up: a cross-sectional study from Egypt

Abstract

Background

The probable association of Toxoplasma gondii (T.gondii) flare-up/co-existence with bacterial meningitis is yet to be elucidated. This study aimed to investigate the possible incidence of T.gondii flare-ups in the cerebrospinal fluid (CSF) suspected of bacterial meningitis as a hidden co-morbid factor. The causative organisms of bacterial meningitis, antibiotic resistance patterns, and physiological aspects of CSF among various age groups were all assessed.

Methods

A cross-sectional study in Almaza Military Hospital, Emergency Department, involved 300 Egyptian patients with symptoms suggestive of meningitis, out of whom 51/ 300 (17.0%) were confirmed by chemical and physiological parameters and microbiological analysis to have bacterial meningitis. Based on age, the patients were divided into Group-1 (< 30 years old) and Group-2 (> 30 years old). The obtained CSF samples were assessed for bacterial growth, antibiotic sensitivity, physiological criteria, and chemical parameters (protein, glucose, and chloride). Toxoplasma was detected using both immune and molecular assays.

Results

Overall, 51 (17%) out of 300 patients were confirmed for bacterial meningitis. Males constituted 66.7%. Group-1 consisted of 41 patients (80.4%) distributed as infants (n = 30, 58.8%), children (n = 7, 13.7%), and neonates (n = 4, 7.8%) (P < 0.001) (males 63.4% and females 36.6%). Group-2 involved 10 patients (19.6%), (males 80% and females 20%). Overall, 49% of the patients received empirical antibiotics, and bacterial growth was present in 51% of the cases. Enterobacter spp. was the most prevalent type of bacteria (15.7%), whereas Pseudomonas and Acinetobacter spp. were the least (2% each). Neonates and children (in subgroup-1) predominantly showed Methicillin-resistant Staphylococcus aureus. 73.7% of infants (subgroup-1) and 80% of group-2 had multidrug-resistant bacteria. Bacterial growth was associated with higher neutrophil count and lower glucose and chloride levels. In group-1, females had a significant increase in neutrophils. The CSF glucose was negatively correlated with neutrophils (r=-0.467) and positively correlated with chloride (r = 0.4). The CSF protein level was positively correlated with neutrophils (r = 0.308), while the chloride level was negatively correlated with neutrophils (r=-0.416) and protein (r = -0.601). The anti-Toxoplasma IgG was positive in 23.5% of cases, indicating exposure to Toxoplasma gondii. All patients were negative for anti-Toxoplasma IgM and the Repeat element (RE) gene. Chronic cerebral toxoplasmosis was higher in the female patients (P = 0.0016).

Conclusions

Despite the variable demographic data, bacterial species, antibiotic resistance, and altered CSF physiological and chemical parameters, toxoplasmosis flare-ups and bacterial meningitis lacked association.

Background

Meningitis can be caused by various pathogens, including bacterial, viral, mycobacterial, parasitic, and fungal infections [1, 2]. Streptococcus pneumoniae, Neisseria meningitidis, Hemophilus influenzae type B, Streptococcus agalactiae group B, and Listeria monocytogenes are the most commonly reported bacterial causes of meningitis globally [3, 4]. The pathological process of bacterial meningitis typically consists of the following stages: invasion of the central nervous system (CNS) and meninges; microbial invasion of the mucous membrane and the intravascular space; microbial multiplication and bacteremia; and translocation over the blood–brain barrier (BBB) [5]. Aging impairs the BBB because it lowers glucose and oxygen levels and changes the body’s ability to eliminate harmful byproducts and interstitial solutes [6, 7].

Despite the cumulative novel diagnostic methods, the preliminary analysis of the chemical and physiological parameters of the cerebrospinal fluid (CSF) remains a critical clinical skill before initiating life-saving therapies, antibiotics, and dexamethasone. In bacterial meningitis, the turbidity and the increased leukocyte count are rarely present on initial CSF analysis. Within the first 48 h, the initial neutrophilic predominance typically turns into lymphocytic. The CSF protein usually increases in bacterial meningitis due to inflammation and increased permeability of the BBB. The low glucose and chloride levels in CSF are other supportive bio-indicator of bacterial meningitis [8].

It is still unclear whether bacterial meningitis could trigger other dormant infections. The obligatory intracellular parasite Toxoplasma gondii is widely distributed worldwide and can infect warm-blooded hosts. Consuming raw/undercooked meat, food, or water that contains oocysts can result in human infection [9]. The range of Toxoplasma IgG seropositivity in Egypt was reported to be 3–42.5%. Moreover, the prevalence of the infection among blood donors was found to be 33.7–67.4% [10]. Although Toxoplasma can enter the CNS through the choroid plexus’s fenestrated endothelium, it is still unknown how Toxoplasma could contribute to meningoencephalitis. Toxoplasmosis has been associated with a variety of brain disorders, from dementia and schizophrenia in chronic infections to life-threatening meningitis and encephalitis in immunocompromised patients [11–16].

Toxoplasma gondii-specific IgM antibodies indicate recent infection within the first 3–6 months, while Toxoplasma IgG antibodies denote past infection [17, 18]. Polymerase chain reaction (PCR) is used to detect T. gondii infection as it efficiently amplifies the DNA material from small samples, particularly in immunosuppressed patients [19].

The objective of this study was to investigate the possibility of T. gondii flare-ups in the CSF of Egyptian patients who may have septic meningitis as a concealed co-morbid condition. The pathogens responsible for bacterial meningitis, the pattern of their antibiotic resistance, and the physiological features of CSF in different age groups were all evaluated.

Methods

Study design and population

The current study is a comparative, cross-sectional study carried out on 300 Egyptian patients suspected of meningitis. It started in April 2024 and terminated in February 2025. Patients involved in the study attended the emergency department, outpatient clinic, or were admitted into the wards of the internal medicine or pediatrics, and were suspected to suffer bacterial meningitis at Almaza Military Hospital, Cairo, Egypt. The diagnosis of meningitis was confirmed by the clinical signs and symptoms, the CSF pressure > 20 cm H₂O, and the initial physical appearance and chemical parameters of the obtained CSF samples. The clinical presentation involved headache, fever or hypothermia, neck stiffness, irritability or lethargy, hypotonia, feeding intolerance or vomiting, respiratory distress, apnea, bradycardia, hypotension, seizures, bulging anterior fontanel, nuchal rigidity, jaundice, hypo- or hyperglycemia, and diarrhea. Exclusion criteria involved pregnancy, autoimmune disease, malignancy, or those who suffer from TB or HIV. Based on age, the patients were divided into two groups (above and below age 30 years).

CSF specimen collection and transportation

A physician used a lumbar puncture to obtain CSF specimens (5–10 ml), which were then fast-tracked to the Microbiology Laboratory for examination within 30 min. Following the guidelines for CSF analysis, each obtained CSF sample was split into three test tubes for chemical analysis, microbiological, and cytological assessments [20].

Microbiological examination

Bacteriological assessment

The volume, color, and appearance of the CSF were recorded as soon as the CSF sample was received at the Microbiology Lab. The centrifuged CSF sediment was plated on blood agar (Oxoid), chocolate agar, MacConkey (Oxoid), and Sabouraud Dextrose agar (Oxoid) plates. The plates were then incubated for 24–48 h at 37 °C under aerobic conditions with 5% CO₂. Using freshly prepared Gram stain reagents, the staining was concurrently done. To identify tubercular meningitis, each CSF sample was further evaluated using the Ziehl-Neelsen stain. Cryptococcal meningitis was identified using a prepared Indian ink.

Isolation and identification of bacteria

Following incubation of the inoculum at 37 °C, the colonies were detected on blood agar, chocolate agar, MacConkey, and Sabouraud Dextrose agar culture plates. Based on their appearance, culture characteristics, and biochemical testing, all positive CSF cultures were identified and described. A series of biochemical assays, including catalase, coagulase, Triple Sugar Iron slant agar (TSI) (Oxoid), urease, lysine decarboxylase (Oxoid), and indole tests (Oxoid) were used to identify Gram-positive and Gram-negative organisms. Motility, Indole, Ornithine test (MIO) (Oxoid) was also performed.

Assessment of antibiotic sensitivity

The Clinical and Laboratory Standards Institute (CLSI) recommends using the standard disk diffusion technique of the modified Kirby-Bauer method for the antibiotic susceptibility test. The inoculum’s opacity was modified to meet the McFarland criteria of 0.5.

Mueller Hinton agar (MHA) (Oxoid) was used to test the antibiotic resistance patterns of the bacterial isolates against widely used antibiotics. The isolates and antibiotic discs were inoculated on MHA according to the CLSI guidelines. Following a 24-hour incubation period at 37 °C, the plates’ findings were recorded (mm) [21]. Isolates that were resistant to three or more classes of antimicrobials were considered as MDR isolates [22] and were selected for further study.

Evaluation of CSF physiological and chemical parameters

• AQuantification of neutrophils in CSF

  Cytological analysis of the CSF sample was conducted on the third test tube. Cell counting was directly done on the sample using a counting chamber. Cell counting was done in duplicate. The CSF sample was checked for turbidity and leukocytosis (usually of polymorphonuclear (PMN) leukocytes). (If the leukocyte count exceeded 100 cells/mm³, differential counting was done in the Hematology unit).

• BQuantification of CSF protein, glucose, and chloride

  CSF aspects used for diagnosing bacterial meningitis were evaluated regarding the cut-off points: protein concentration (15 to 60 milligrams per deciliter (mg/dL), glucose concentration (50 mg/dL), and chloride level (113 - 130 mEq/L). CSF total protein was quantitatively analyzed using the benzethonium chloride turbidimetric method on the Cobas® 6000 analyzer series, module C503 (Roche Diagnostics Corporation, Indianapolis, IN, USA; manufactured by Hitachi High-Technologies Corporation, Ibaraki, Japan). CSF glucose was measured using the hexokinase method on the same analyzer (module C503). CSF chloride levels were determined using the ion-selective electrode method on the Cobas ® 8000 analyzer series, module C311 (Roche Diagnostics Corporation, Indianapolis, IN, USA; manufactured by Hitachi High-Technologies Corporation, Ibaraki, Japan).

Parasitological assessment

Detection of T. gondii antibodies

All CSF samples were initially evaluated for the existence of IgG and IgM anti-Toxoplasma gondii. The OnSite Toxo IgG/IgM Combo Rapid Test for T. gondii (CTK BIOTECH, Cat No R0234C, USA) was used.

Genomic DNA extraction and PCR amplification of T. gondii

The DNA was extracted from CSF samples using the Thermo Scientific™ K0171 kit per the manufacturer’s instructions. These DNA samples were stored at −20̊C. NanoDrop^TM 2000/2000c was used to evaluate the concentration and purity of DNA (Thermo Fisher Scientific, Waltham, MA, USA). Conventional PCR for T. gondii targeting the 529-RE (repetitive element) was performed in a final volume of 25 µl. The primer sequences were as follows; Forward (Tox-4): 5’-CGCTGCAGGGAGGAAGACGAAAGTTG-3’ and Reverse (Tox-5): 5’- CGCTGCAGACACAGTGCATCTGGATT-3’ [20]. The PCR amplification was performed under the following conditions: 5 min of initial denaturation at 95̊ C; 35 cycles of 60 s at 94̊ C, 60 s at 55̊C, and 60 s at 72̊C; and a final elongation step at 72̊ C for 7 min. The PCR product was analyzed with 1.5% agarose gel electrophoresis.

Statistical analysis

The statistical software for the social sciences (SPSS) version 28 (IBM Corp., Armonk, NY, USA, 2021) was used to code and enter the data. For quantitative data, the median and interquartile ranges were used; for categorical data, frequency (count) and relative frequency (%) were used to summarize the data. Non-parametric Mann-Whitney and Kruskal-Wallis tests were used to compare quantitative variables. We used the Chi-square (X²) test to compare categorical data. When the expected frequency is less than five, an exact test was utilized. The Spearman correlation coefficient was utilized for establishing correlations between quantitative variables. There are four categories for the correlation coefficient (R-value): very weak (0-0.19), weak (0.2–0.39), moderate (0.40–0.59), strong (0.6–0.79), and very strong (0.8-1). The positive sign (+ ve) denotes positive correlation, whereas the negative sign (-ve) indicates an inverse relationship. P-values less than 0.05 were considered statistically significant.

Results

Demographic characteristics of study participants

Out of the 300 cases, a total of 51 patients (17%) with confirmed bacterial meningitis were enrolled in the current study. Out of these cases, 34 (66.7%) were males and 17 (33.3%) were females (P < 0.05). Group 1 (below the age of 30) included 41 patients (80.4%), whilst only 10 (19.6%) patients represented Group 2 (above age 30) (P < 0.001). In Group 1, the majority of the patients were infants (n = 30, 58.8%), children (n = 7, 13.7%), followed by neonates (n = 4, 7.8%) (P < 0.001). A full description of the patients’ age and sex is demonstrated in Tables 1 and 2, respectively.

Table 1.

Incidence of clinically presented bacterial meningitis

Age group		Median	1st quartile	3rd quartile
Group 1	Neonate’s age (days)	22.5	20.5	24
	Infant’s age (months)	3	2	6
	Children’s age (years)	3	1.5	5
Group 2	Adult’s age (years)	48	30	62

Table 2.

Distribution of sex among patients

Sex	Females		Group1	Group 2	Total
		Count	15a	2a	17
		% within sex	88.2%	11.8%	100.0%
		% within group	36.6%	20.0%	33.3%
		% of Total	29.4%	3.9%	33.3%
	Males	Count	26a	8a	34
		% within sex	76.5%	23.5%	100.0%
		% within group	63.4%	80.0%	66.7%
		% of Total	51.0%	15.7%	66.7%

Each subscript letter denotes a subset of group categories whose column proportions do not differ significantly from each other at the 0.05 level

Microbiological characteristics of CSF

Among the tested CSF samples, 25 samples (49%) were gathered after the primary dose of empirical antibiotic treatment, and 26 samples (51%) were recruited before the administration of antibiotics. Of the total collected CSF samples, 40 samples (78.4%) were physically turbid during collection, 7 (13.7%) were semi-turbid, and 4 (7.8%) were clear. Neither the Ziehl-Neelsen stain nor the Indian ink preparation revealed the presence of acid-fast bacilli nor Cryptococcus fungal pathogens, respectively. None of the patients who had previously received empirical antibiotics revealed the presence of bacterial growth.

Prevalence of bacterial growth

The overall count of samples positive for bacteria in this study was 26 (51%). There was no significant difference in the age and sex of the participants with CSF samples positive for bacterial growth (Table 3). The prevalence of Gram-negative and Gram-positive bacteria yielded insignificant differences (P = 0.157). Overall, the bacterial isolates from the tested CSF samples were as follows; Enterobacter spp. (15.7%, 8/26), Klebsiella spp. (13.7%, 7/26), Staph. aureus (7.8%, 4/26), Coagulase negative staph. (CoNS) (7.8%, 4/26), E. coli (2%, 1/26), Acinetobacter spp. (2.0%, 1/26) and pseudomonas aeruginosa (2.0%, 1/26). In Group 1, CSF of infants aged less than 2 years mainly revealed Klebsiella spp. (23.3%, 7/19), Enterobacter spp. (20.0%, 6/19) and (CoNS 13.3%, 4/19). However, in Group 2, a higher rate of growth of Enterobacter spp. (20.0%, 2/5) was observed (Table 4).

Table 3.

Incidence of bacterial growth relative to age and sex

			Positive CSF culture (n = 26)	Negative CSF culture (n = 25)	P value
			N (%)	N (%)
Age	Group 1	Neonates	1 (3.8%)	3 (12.0%)	0.079
		Infants	19 (73.1%)	11 (44.0%)
		Children	1 (3.8%)	6 (24.0%)
	Group2	Adult	5 (19.2%)	5 (20.0%)
Sex		Female	9 (34.6%)	8 (32.0%)	0.843
		Male	17 (65.4%)	17 (68.0%)

Table 4.

Incidence rate of the bacterial isolates in group 1 and group 2

		Group 1			Group 2
		Neonates	Infants	Children	Adult
		N (%)	N (%)	N (%)	N (%)
Bacterial isolates	CoNS	0 (0%)	4 (13.3%)	0 (0.0%)	0 (0.0%)
	S.aureus	1 (25%)	1 (3.3%)	1 (14.3%)	1 (10.0%)
	P.aeruginosa	0 (0%)	1 (3.3%)	0 (0.0%)	0 (0.0%)
	Klebsiella spp.	0 (0%)	7 (23.3%)	0 (0.0%)	0 (0.0%)
	Enterobacter spp.	0 (0.0%)	6 (20.0%)	0 (0.0%)	2 (20.0%)
	E.coli	0 (0.0%)	0 (0.0%)	0 (0.0%)	1 (10%)
	Acinetobacter spp.	0 (0.0%)	0 (0.0%)	0 (0.0%)	1 (10%)

Antibiotics sensitivity pattern

Antibiotic susceptibility tests were done for all bacterial isolates. All S. aureus isolates (100%, 4/4) were resistant to Methicillin (MRSA). However, all those MRSA isolates demonstrated high sensitivity to both vancomycin (100%, 4/4) and linezolid (100%, 4/4). All the CoNS isolates (100%, 4/4) were resistant to Methicillin (MR) and susceptible to both vancomycin and linezolid. All the Enterobacter spp. isolates were MDR, but they were susceptible to tigecycline (TGC). Only one Enterobacter isolate (12.5%, 1/8) was susceptible to amikacin (AK). All the Klebsiella spp. isolates were MDR, but they were highly susceptible to tigecycline (85.7%, 6/7), and only one isolate was susceptible to both imipenem (IPM) and meropenem (MEM) (14.3%, 1/7). The only E. coli isolate was MDR, but it demonstrated high susceptibility to amikacin (AK). The only Acinetobacter spp. isolate was MDR, but it demonstrated high susceptibility to tigecycline. The only Pseudomonas aeruginosa isolate was MDR, but it demonstrated high susceptibility to both amikacin (AK) and tazobactam (TZP). The distribution of antibiotic resistance in Groups 1 & 2 is shown in Table 5.

Table 5.

Pattern of antibiotic resistance in group 1 and group 2

		Group 1			Group 2
		Neonates	Infants	Children	Adult
		N (%)	N (%)	N (%)	N (%)
The pattern of antibiotic resistance	MRSA	1 (100%)	1 (5.3%)	1 (100%)	1 (20%)
	MR CONS	0 (0.0%)	4 (21.1%)	0 (0.0%)	0 (0.0%)
	MDR	0 (0.0%)	14 (73.7%)	0 (0.0%)	4 (80%)

MRSA: Methicillin-resistant Staphylococcus aureus, MR: Methicillin-resistant, CoNS: Coagulase-negative Staphylococci, MDR: Multidrug-Resistant

Physiological and chemical parameters of CSF with age and sex

The CSF characteristics showed insignificant differences between the age groups, however, Group 1 demonstrated higher values of neutrophil counts and total protein concentrations (40 cells/µl and 960 mg/dl, respectively). The median value of CSF glucose in Group 1 was 37 mg/dl, and in Group 2 was 44 mg/dl (P = 0.953). Also, the CSF chloride scored relatively the same values in Group 1 and Group 2, as shown in Table 6.

Table 6.

Distribution of various CSF characteristics with age groups

	Group 1			Group 2			P value
	Median	1 st quartile	3rd quartile	Median	1 st quartile	3rd quartile
Neutrophils (/mm3)	40	10	600	26	10	500	0.534
Glucose (mg/dL)	37	9	53	44	0.50	86	0.953
Total protein (mg/dL)	960	46	1461	210.50	15	580	0.129
Chloride (mEq/L)	108	99	120	108.50	102	120	0.678

In Group 1, there was a significant increase in the neutrophil counts in the females relative to the males (P = 0.037). In Group 2, none of the CSF parameters appeared to be affected by the patients’ sex, as shown in Table 7.

Table 7.

Distribution of various CSF characteristics by sex

Groups	CSF parameters	Sex						P value
		Female			Male
		Median	1 st quartile	3rd quartile	Median	1 st quartile	3rd quartile
Group1	Neutrophils (/mm3)	400	40	1100	32.50	10	310	0.037
	Glucose ( mg/dL)	40.50	5	53	27.50	9	56	0.883
	Total protein ( mg/dL)	960	36	2149	912	108	1405	0.968
	Chloride (mEq/L)	110	99	123	105.50	98	120	0.429
Group 2	Neutrophils (/mm3)	6755	10	13,500	26	10	275	0.711
	Glucose ( mg/dl)	45.25	0.50	90	44	2.75	79.50	0.711
	Total protein (mg/dl)	296	12	580	210.50	26.50	401.50	0.711
	Chloride (mEq/L)	102.00	92.00	112.00	108.50	104.00	122.50	0.533

Physiological and chemical parameters of CSF with bacterial growth

CSF with proven bacterial growth showed significantly-higher CSF neutrophil counts and lower glucose and chloride levels when compared with the suspected patients who had no apparent growth of bacteria on culture (Table 8).

Table 8.

The diagnostic performance of various CSF characteristics with bacterial growth

	Positive CSF culture			Negative CSF culture			P value
	Median	1 st quartile	3rd quartile	Median	1 st quartile	3rd quartile
Neutrophils (/mm3)	475	40	800	12	10	40	< 0.001
Glucose ( mg/dL)	11	2.90	40	54	30	80	0.001
Total protein ( mg/dL)	735	210	1318	193	20	1405	0.274
Chloride (mEq/L)	103	92	116	112	107	120	0.012

Interactions of various physiological and chemical parameters

The CSF glucose revealed negative correlation with the neutrophil count (r=−0.467-, P = 0.001). The CSF glucose and the CSF chloride levels showed moderate correlation (r = 0.4, P = 0.005). There was no significant association between the CSF glucose and the CSF total protein concentration (r=−0.275, P = 0.051). Neutrophils’ count and CSF protein concentration displayed significant correlation (r = 0.308, P = 0.028). Nevertheless, a moderate inverse relationship was found between the Neutrophils’ count and the CSF chloride level (r =−0.416, P = 0.002). The CSF chloride level also exhibited a strong inverse association with increased protein level (r = −0.601, P < 0.001) (Fig. 1A-E).

Fig. 1.

Correlation analysis between the CSF physiological parameters

The overall prevalence of T. gondii infection in CSF

Among the 51 enrolled patients, 23.5% of the cases (12/51) tested positive for anti-Toxoplasma IgG antibodies in their sera, indicating exposure to Toxoplasma gondii, while no cases showed positivity for anti-Toxoplasma IgM antibodies. None of the samples of patients could amplify the Toxoplasma RE gene The relationship between the seroprevalence of anti-Toxoplasma IgG antibodies and their association with various demographic variables (age and sex) is illustrated in Table 9.

Table 9.

Incidence of T. gondii IgG relative to age and sex in CSF

Variables			Positive T. gondii IgG (n = 12)	Negative T. gondii IgG (n = 39)	P-value
			N (%)	N (%)
Age	Group 1	Neonates	0 (0.0%)	4 (10.2%)	0 0.379
		Infants	0 (0.0%)	30 (76.9%)
		Children	3 (25.0%)	4 (10.2%)
	Group2	Adult	9 (75.0%)	1 (2.7%)
Sex		Female	9 (75.0%)	8 (20.5%)	0.0016
		Male	3 (25.0%)	31 (79.5%)

The prevalence of chronic toxoplasmosis was found to be significantly higher in female than in male patients (P = 0.0016) (Table 9).

Discussion

The most fatal disease that seriously endangers life is still bacterial meningitis. Despite the great advances in supportive care and antibiotic therapy, bacterial meningitis remains one of the most fatal CNS infections. According to World Health Organization (WHO) estimates, meningitis infections are widespread in Egypt, where there are 135–200,000 fatal cases per year out of around 1 million cases globally [23]. The current investigation sought to investigate the incidence of T. gondii flare-ups in the CSF as a potential hidden co-morbid condition. This included evaluating the organisms responsible for bacterial meningitis, the pattern of antibiotic resistance, and the chemico-physiological aspects of CSF in the different age groups.

The majority of patients with bacterial meningitis were males (66.7%). This result is close to that reported by Moradi et al., [24], who found that 59.6% of patients with meningitis were men. Males were more likely to develop bacterial meningitis; this could be due to the sex hormones that have been found to affect the efflux transport at the BBB, the nutrient uptake, and the tight junction integrity. Such effects influence the administration of CNS-targeted drugs and brain homeostasis [25].

The incidences of bacterial meningitis in patients below the age of thirty (group 1) were higher than in group 2 (above thirty). This aligns with a similar study done in Egypt, which identified children as the most susceptible age group for bacterial meningitis. The higher susceptibility of early-age groups to bacterial infection is due to the qualitative and quantitative deficits of the innate immune system [23].

The current study also recorded a relative increase in the CSF protein and neutrophil counts of the meningitis cases. Tan et al., [26] demonstrated that the persistence of these indicators is related to poor outcomes.

The prevalence of Gram-negative and Gram-positive bacteria in the current study yielded insignificant differences. Yet, in Group 1, patients less than 30 years, Klebsiella spp., Enterobacter spp., and CoNS were the most prevalent bacterial species. While in Group 2, there was a higher growth rate of Enterobacter spp. Similar studies conducted in developing and underdeveloped countries reported the prevalence of these pathogenic strains as the dominant cause of infections in CSF [27]. Prior studies in Egypt determined Streptococcus pneumoniae as the leading cause of bacterial meningitis among adults, whereas Neisseria meningitides was the second or third leading cause [28]. This reflects the dynamic change in the epidemiology of the disease, where Neisseria meningitidis. was for a long time the main etiological agent.

Bacterial meningitis in infants less than 2 years old represented the highest proportion of cases. Consistent with these results, Ali et al., [29] showed a significantly high percentage of bacterial meningitis in children (less than one year of age), in which E. coli and K. pneumoniae were the most prevalent etiologic agents. Likewise, a study conducted on the CSF of Chinese children revealed the prevalence of E. coli in 28.5% of the cases [27].

The antimicrobial resistance in all the isolated bacterial pathogens from the CSF of bacterial meningitis patients was evaluated in the present study. All the Gram-negative isolates have demonstrated MDR. Also, Staphylococci, despite showing significant susceptibility to vancomycin and linezolid, have demonstrated methicillin resistance, especially with the S. aureus and the CoNS species. The infection rate among individuals with bacterial meningitis has recently increased due to the growing resistance to third-generation antibiotics [30]. The pathogenic E. Coli and Klebsiella species isolated from several hospitalized patients were also found to be highly resistant to first-generation (ampicillin 91%, cefixime 100%, and amoxicillin 85%) as well as second- and third-generation (cefotaxime 82% and cefaclor 100%) antibiotics [31].

Tigabu et al., [32] found that adult patients with bacterial meningitis had significant S. aureus resistance (66.80%) to both amoxicillin and erythromycin. High resistance of S. aureus to amoxicillin (100%) has also been previously recorded [33].

Neutrophil counts were significantly higher in female patients below their thirties compared with the age-matched males. Estradiol has been previously shown to positively correlate with the C-reactive protein, proinflammatory cytokine levels, CSF protein, and the severity of the disease in bacterial meningitis [34].

Correlations between various parameters of the CSF were consistent with the diagnostic indicators of bacterial meningitis [35]. Yet, regarding the current inverse relationship between CSF chloride and protein levels, Fang et al., [36] deduced that CSF chloride changes in relation to the plasma chloride levels. When the osmolarity of the CSF is kept in equilibrium, an increase in protein concentration induces a decrease in the chloride level. In addition, bacterial infections in the CNS can cause a reduction in the CSF chloride level.

Despite the aforementioned variations in the demographic data of the participating subjects, altered CSF characteristics, and the variances in bacterial growth, Toxoplasma Ig M and the RE gene were not detected throughout the study. This might be similar to Schluter and Barragan [37], who deduced that cerebral toxoplasmosis often manifests as encephalitis, whereas meningitis is rare. The negative T. gondii IgM-DNA (RE gene) profile in the current study also ruled out acute and recent infections [38, 39].

The T. gondii IgG was detected in 23.5% of the CSF specimens. This result is consistent with the results of a previous review reporting that the sero-prevalence of T. gondii IgG among the general Egyptian population ranges from 3 to 42.5%, depending on the geographical area, socioeconomic status, and exposure to the parasitic infection [40]. Similarly, a study from Iraq showed a comparable seroprevalence rate of chronic toxoplasmosis [41]. Also, a Tunisian study demonstrated that the seroprevalence of T.gondii IgG was 44% [42].

The current study also reported a negative Toxoplasma immunoglobulin-DNA (RE gene) profile in all the studied cases, which rules out the immune-compromised status in the studied cases. A previous study by Hajizadeh et al. [43] reported increased prevalence of T. gondii IgM and DNA in malignancy compared with healthy status. The Toxoplasma-GRA6 gene was also recently detected in the CSF from HIV/AIDS patients [44]. In addition, increased seroprevalence of IgG was reported among Egyptian patients with solid and hematological cancers (approximately 77.3% and 46.4%, respectively) [45]. A prior meta-analysis showed that immunosuppressed patients, such as HIV/AIDS, cancer, and transplant recipients, have a pooled prevalence of T. gondii-IgG [46]. Moawad et al., [47] also reported higher Toxoplasma seropositivity in hemodialysis Egyptian children (28%) than their controls (16%).

The Toxoplasma profile (immunoglobulins and the RE gene) did not show any positive results in the CSF from neonates and infants. This excludes maternal infection in the third trimester or transplacental transmission in these patients [48]. CSF-Toxoplasma IgG in females was significantly higher than in males, which might reflect their higher exposure (e.g., occupational, contact with cats, and/or contaminated soil) [49]. Similarly, Mahmoud et al. [50] reported increased prevalence of toxoplasmosis among Libyan women.

Olivera et al., [51] determined that Toxoplasma acts differently with the cerebral vasculature compared with other organs. In experimental models, the number of tachyzoites in the cortical capillaries of the brain appeared lower than those detected in the pulmonary and hepatic vasculature. Thus, these findings propose the high resistance of the BBB to the adhesion, invasion, and translocation of the parasite. Ross et al., [52] showed that the passage of T. gondii to the CNS requires particular disruption of the focal adhesion kinases in the endothelial lining to increase the vascular permeability of the BBB. The infiltration of the inflammatory leukocytes occurs after 10 to 15 days of infection, with an assumed contribution to the translocation of the Toxoplasma.

Conclusion

This study revealed that bacterial meningitis had a higher incidence in males and appeared to be more common at young ages (< 30 years), particularly in infants. It was also noted that the empirical administration of antibiotics, in turn, hampered bacterial growth. The Gram-negative bacteria like K. pneumoniae and Enterobacter spp. infections showed higher incidences, whilst S. pneumoniae, Haemophilus influenza, and Neisseria meningitides infections were particularly absent. Multi-drug resistance in the clinical isolates was also notable, wherein empirical administration of antibiotic therapy seemed to be one of the causes. Altered CSF physiological indices and their interactions confirmed the diagnosis of bacterial meningitis. Despite the aforementioned microbiological and physiological indices, the Toxoplasma IgM antibody and Toxoplasma-RE gene were not detected throughout the study. Yet, anti-Toxoplasma IgG in CSF showed positivity in 23.5% of cases, indicating past exposure to the parasitic infection. CSF-Toxoplasma IgG was significantly higher in females. These findings rule out the probability of an association between Toxoplasma flare-up and bacterial meningitis. Further studies are recommended on a wider scale of patients.

Abbreviations

BBB

Blood–brain barrier

CSF

Cerebrospinal fluid

CNS

Central nervous system

PCR

Polymerase chain reaction

Authors’ contributions

R.Y.S., B.E.A. and M.F.F. collected the data. R.Y.S., B.E.A., M.A.A. A.I. and N.Y.S. analysed and interpreted the data. A.I., R.Y.S. and E.A.E. performed the practical analysis. A.I. and E.A.E. wrote the original draft of the manuscript. All authors shared in reviewed, edited and approved the final version of the manuscript.

Funding

The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University for funding this work through Large Research Project under grant number RGP2/430/46.

Data availability

‘’The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Al-Cairo alainy faculty of medicine, Cairo University.‘’.

Declarations

Ethics approval and consent to participate

Ethical clearance for the study was obtained from the Armed Forces College of Medicine, Cairo, Egypt (reference number: 465, date: 20-4-2024). All procedures involved in the study were under the ethical consideration of the National Research Committee and with the 1964 Helsinki Declaration and its succeeding amendments or comparable ethical standards. Permission to conduct this study and ethical approval were received from the IRB Vice Chair and IRB Chair, Armed Forces College of Medicine, Cairo, Egypt. The present study was conducted only on CSF specimens of the bacterial meningitis suspected cases. Informed consent was obtained from all participants, by adult participants or legal guardians of children.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.