Bacterial meningitis (BM) is a life-threatening condition which affects the central nervous system (CNS). Its incidence rate is estimated between 0.22 - 2.66 and 0.81 - 6.1 cases/1000 lives worldwide and in Africa respectively. The objective of this study was to determine the frequency of bacterial organisms isolated from CSF of children less than five years old in Windhoek. A retrospective analysis was performed on 784 results of CSF submitted to the Namibia Institute of Pathology (NIP) from January 2010 to August 2014. The results showed that out of the suspected meningitis cases, 18 (28.6%) were due to Streptococcus pneumoniae, making it the frequently isolated organism, followed by Staphylococcus aureus 7 (11.1%), Escherichia coli 5 (7.9%) and Haemophilus influenzae 4 (6.3%). Streptococcus pneumoniae showed high resistance to penicillin 17 (70.5%) & cotrimoxazole 16 (93.7%) and 100% susceptibility to ciprofloxacin (5), vancomycin (13) & ceftri-axone (8). Haemophilus influenzae showed moderate resistance to cotrimoxazole 3 (66%) & tet-racycline 2 (50%). It showed 100% sensitivity to chloramphenicol (4), cefuroxime (2) & ceftriaxone (3). Neisseria meningitidis showed high resistance to cotrimoxazole by 100% (n = 2) and high sensitivity to chloramphenicol (n = 2), ceftriaxone & penicillin by 100% (n = 2). Streptococcus agalactiae was resistant to tetracycline and sensitive to penicillin & erythromycin by 100% (n = 1). Streptococcus pneumoniae was isolated the most in this current study and it had high resistance to penicillin & cotrimoxazole. There was a significant difference between results CSF culture and PCR, Gram stain, CSF cell count, protein & glucose, as all comparisons yielded in P values less than 0.05, indicating a significant statistical association.
Meningitis is a result of the intrusion of the meninges by microbial agents. BM claims many lives annually and it is associated with a high incidence of disability and long-term health implications. Furthermore, about 5% mortality rates have been reported in children from developed countries, whereas in developing countries, the rates have been estimated at 30% [
The development of BM usually follows colonization of the respiratory mucosa by invasive bacteria. This mechanism involves attachment of the bacterial organism onto the nasopharyngeal mucosal cells, colonization of the respiratory mucosa, invasion of the intravascular space, bacteraemia, meningeal invasion, bacterial replication in the subarachnoid space, thus leading to inflammation of the subarachnoid space [
The main bacterial causes of meningitis in neonates (premature and full term) up to 3 months of age include S. agalactiae or group B streptococcus (GBS), coliform bacilli such as E. coli possessing K1 antigens, as well as L. monocytogenes [
Clinical manifestations of bacterial meningitis include fever, headache, hypothermia, nausea, vomiting, photophobia, confusion, lethargy, irritability, poor feeding, respiratory distress, diarrhoea, bulging fontanelles and seizures [
In Namibia in 2003, nine cases of BM were reported in neonates less than 1 month, 93 cases in infants aged 1 - 11 months & 80 cases in children aged 1 - 5 years. It was reported that Extended Spectrum Beta Lactamase (ESBL) Klebsiella pneumonia was most commonly isolated from CSF collected from neonates. Streptococci, H. influenzae and Staphylococci were commonly isolated from CSF collected from infants aged 1 - 11 months. N. meningitidis, H. influenzae and S. pneumoniae were mostly isolated from children aged 1 - 5 years [
The study was aimed at investigating the frequency of bacterial organisms isolated from children less than 5 years in Windhoek for the period 2010 to 2014, as well as their antimicrobial susceptibility patterns.
This research was a descriptive retrospective study, conducted to establish the frequency of bacterial organisms isolated from CSF samples of children under the age of five years in Windhoek, Namibia from the year 2010 to 2014. The study included 784 CSF results of children under the age of five years whose CSF samples were examined at the Namibia Institute of Pathology central laboratory in Windhoek, Namibia, from 2010 to 2014. These were children admitted at the Katutura intermediate hospital and Windhoek central hospital. The Katutura intermediate hospital is the referral centre for all hospitals across Namibia and has a high admission rate due to referred cases. The Windhoek central hospital is classified as the national referral hospital handling specialised cases referred from the Katutura hospital and from the whole of Namibia.
All bacterial culture positive and negative results were included in the study. CSF results which were positive for fungal, parasitic or viral pathogens were excluded from the study. Follow-up results and results that queried contamination were also excluded from the study. This study was carried out in Windhoek, Namibia.
CSF samples were examined at NIP microbiology laboratory using a procedure cited in Cheesbrough, 2006, which includes macroscopy, cell count, culture, Gram and methylene blue stain, protein and glucose quantitation. CSF culture was performed on 5% sheep blood agar and chocolate agar. Blood agar was made using Columbia blood agar base (Oxoid, United Kingdom) and 5% sheep blood, whereas chocolate agar comprised of the same components, with Hemin (factor X) and Nicotinamide adenine dinucleotide (NAD or V factor) released from haemolysed blood [
Antimicrobial susceptibility testing was done manually from 2010 to 2013 using the Kirby-Bauer disc diffusion method, and on the VITEK® 2 (bioMérieux, United States of America) in 2014. Only the antibiotics most commonly used in empirical therapy of meningitis were included in the study, based on the Namibia Standards Treatment Guideline. These were: penicillin, ceftriaxone, chloramphenicol, vancomycin, erythromycin, clindamycin, ciprofloxacin, cotrimoxazole and tetracycline. A bacterial isolate was defined sensitive, intermediate sensitivity or resistant using Clinical Laboratory Standards Institute (CLSI) guidelines on antimicrobial susceptibility [
Data was analyzed using IBM SPSS version 22. Descriptive statistics were used to summarize the frequencies of bacterial organisms isolated from CSF, and their susceptibility patterns to various antimicrobial agents. The frequency of bacterial organisms causing meningitis in children was determined for the period of 2010 to 2014 and displayed in a frequency table. The percentage sensitivity and resistance of bacterial isolates were calculated using all the results from 2010 to 2014. Inconclusive results, reports with missing data and outliers were excluded from all statistical analysis.
The comparison of culture, PCR, Gram stain, cell count, protein and glucose was performed using chi-square and a P value less than 0.05 was considered significant. The comparison of culture and PCR was done using 2014 data only, while the comparison of culture, Gram stain, cell count, protein and glucose was done on 2010 to 2014 data. All comparison results were displayed in contingency tables.
Since the study was conducted on de-identified electronic results of CSF tested at NIP, individual patient consent was not required. Authorization to conduct this study was granted by the researches committees of the Ministry of Health and Social Services (MoHSS), NIP and NUST.
A total of 784 cerebrospinal fluid samples collected from children below five years were examined for suspected bacterial meningitis at NIP from January 2010 to August 2014. Out of the 784 CSF samples, 63 positive cases were detected, which was a positivity rate of 8%.
S. pneumoniae showed high resistance to penicillin and cotrimoxazole, as well as moderate resistance to erythromycin, clindamycin and tetracycline. All S. pneumoniae isolates were sensitive to ciprofloxacin, vancomycin and ceftriaxone.
From the 784 CSF samples, 52 were examined using both culture and PCR, and the results were compared. A positive culture result was defined as isolation of any bacterial organism from CSF and a positive PCR result was defined as detection of any bacterial organism in CSF.
From the 784 CSF samples of children under 5 years that were examined from 2010 to 2014 at NIP in Windhoek, 762 had both culture and Gram stain results. The results were compared to determine if there is a significant difference between culture and Gram stain. A Gram stain result was considered positive if bacteria were seen on the Gram-stained smear.
Out of a total of 784 CSF samples, 702 had both the neutrophil count and culture results.
Protein estimates and culture results were obtained in 700 of the total 784 CSF samples examined. Of these 700, 93.1% (n = 652) were culture negative while 6.9% (n = 48) were culture positive.
Glucose estimates and culture results were obtained in 714 of the total 784 CSF samples examined. Culture was negative in 93.8% (n = 670) and positive in 6.2% (n = 44) cases of the total 714 cases.
This study aimed at determining the frequency of bacterial organisms isolated from CSF of children below five years and to determine the antimicrobial sensitivity patterns of the four well known leading causes of bacterial meningitis in infants.
The findings of this current study were different from those obtained from a similar study conducted in Mozambique in 2006, whereby 70.6% of all bacterial organisms isolated from 43 positive cases were H. influenzae, S. pneumoniae and N. meningitidis [
Bacterial organisms isolated | Frequency | Percent (%) |
---|---|---|
S. pneumoniae | 18 | 28.60% |
H. influenzae | 4 | 6.30% |
N. meningitidis | 2 | 3.10% |
S. agalactiae | 1 | 1.60% |
K. pneumoniae | 3 | 4.80% |
E. coli | 5 | 7.90% |
S. aureus | 7 | 11.10% |
Pseudomonas species | 1 | 1.60% |
Staphylococcus hominis (S. hominis) | 2 | 3.10% |
Acinetobactern species | 4 | 6.30% |
Serratia species | 3 | 4.80% |
Enterococcus species | 3 | 4.80% |
Moraxella species | 1 | 1.60% |
Enterobacter species | 1 | 1.60% |
S. epidermidis | 7 | 11.10% |
Sphingomonas paucimobilis (S. paucimobilis) | 1 | 1.60% |
Total | 63 | 100 |
Organism isolates | Total isolates | Antibiograms | Total tested | S | % S | R | % R | I | % I |
---|---|---|---|---|---|---|---|---|---|
S. pneumoniae | 18 | Penicillin | 17 | 5 | 29.4 | 12 | 70.5 | 0 | 0 |
Ceftriaxone | 8 | 8 | 100 | 0 | 0 | 0 | 0 | ||
Chloramphenicol | 18 | 17 | 94.4 | 0 | 0 | 1 | 5.5 | ||
Vancomycin | 13 | 13 | 100 | 0 | 0 | 0 | 0 | ||
Erythromycin | 18 | 14 | 77.7 | 4 | 22.2 | 0 | 0 | ||
Clindamycin | 17 | 15 | 88 | 2 | 12 | 0 | 0 | ||
Ciprofloxacin | 5 | 5 | 100 | 0 | 0 | 0 | 0 | ||
Cotrimoxazole | 16 | 1 | 6.25 | 15 | 93.7 | 0 | 0 | ||
Tetracycline | 18 | 14 | 77.7 | 3 | 16.6 | 1 | 6.25 | ||
S. agalactiae | 1 | Penicillin | 1 | 1 | 100 | 0 | 0 | 0 | 0 |
Erythromycin | 1 | 1 | 100 | 0 | 0 | 0 | 0 | ||
Tetracycline | 1 | 0 | 0 | 1 | 100 | 0 | 0 | ||
N. meningitidis | 2 | Penicillin | 2 | 2 | 100 | 0 | 0 | 0 | 0 |
Ceftriaxone | 2 | 2 | 100 | 0 | 0 | 0 | 0 | ||
Chloramphenicol | 2 | 2 | 100 | 0 | 0 | 0 | 0 | ||
Cotrimoxazole | 2 | 0 | 0 | 2 | 100 | 0 | 0 | ||
H. influenzae | 4 | Ceftriaxone | 3 | 3 | 100 | 0 | 0 | 0 | 0 |
Chloramphenicol | 4 | 4 | 100 | 0 | 0 | 0 | 0 | ||
Cotrimoxazole | 3 | 1 | 33.3 | 2 | 66.6 | 0 | 0 | ||
Tetracycline | 2 | 1 | 50 | 1 | 50 | 0 | 0 | ||
Cefuroxime | 2 | 2 | 100 | 0 | 0 | 0 | 0 |
Abbreviations: S = sensitive, R = resistant, I = intermediate.
Culture results | Total | |||
---|---|---|---|---|
Negative | Positive | |||
PCR results | Negative | 21 (44.7%) | 1 (20%) | 22 (42.3%) |
Positive | 5 (10.6%) | 3 (60%) | 8 (15.4%) | |
Inconclusive | 21 (44.7%) | 1 (20%) | 22 (42.3%) | |
Total | 47 (100%) | 5 (100%) | 52 (100%) |
χ2 value = 8.40 (P value = 0.015).
Culture results | Total | |||
---|---|---|---|---|
Negative | Positive | |||
Gram stain | Negative | 698 (99.3%) | 19 (32.2%) | 717 (94.1%) |
Positive | 5 (0.7%) | 40 (67.8%) | 45 (5.9%) | |
Total | 703 (100%) | 59 (100%) | 762 (100%) |
χ2 value = 15.14 (P value = 0.0001).
Neutrophil count (mm3) | Total | |||
---|---|---|---|---|
<5 mm3 | >5 mm3 | |||
Culture results | Negative | 558 (98.5%) | 107 (78.8%) | 665 (94.7%) |
Positive | 8 (1.4%) | 29 (21.3%) | 37 (5.3%) | |
Total | 566 (100%) | 136 (100%) | 702 (100%) |
χ2 value = 15.81 (P value = 0.0001).
CSF protein (g/L) | Total | ||||
---|---|---|---|---|---|
Low | Normal | High | |||
Culture results | Negative | 381 (98.7%) | 168 (95.5%) | 103 (74.6%) | 652 (93.1%) |
Positive | 5 (1.30%) | 8 (4.5%) | 35 (25.4%) | 48 (6.9%) | |
Total | 386 (100%) | 176 (100%) | 138 (100%) | 700 (100%) |
χ2 value = 18.42 (P value = 0.0001).
CSF glucose (mmol/L) | Total | ||||
---|---|---|---|---|---|
Low | Normal | High | |||
Culture results | Negative | 128 (81.0%) | 495 (97.6%) | 47 (95.9%) | 670 (93.8%) |
Positive | 30 (18.9%) | 12 (2.4%) | 2 (4.1%) | 44 (6.2%) | |
Total | 158 (100%) | 507 (100%) | 49 (100%) | 714 (100%) |
χ2 value = 18.87 (P value = 0.0001).
Studies have shown that group B streptococcus is the leading causative agent of neonatal meningitis, implicated in up to 50% cases [
S. pneumoniae was the most frequent bacterium isolated from CSF of children below five years in Windhoek. These findings correlate with the findings obtained from a similar study done in Namibia in 2013, whereby S. pneumoniae was most frequently isolated from CSF of children aged between 1 - 11 months from 2009-2012 [
S. pneumoniae showed very high resistance to penicillin and to cotrimoxazole. Furthermore, it exhibited relative resistance to erythromycin and clindamycin ranging from 11.7% to 22.2%, as shown in
On the contrary, a similar study conducted in Mozambique found S. pneumoniae to be susceptible to penicillin [
S. pneumoniae showed high sensitivity to chloramphenicol, which was also demonstrated in a surveillance study conducted in Mozambique, in which it was susceptible to chloramphenicol [
N. meningitidis showed high sensitivity to penicillin and chloramphenicol as shown in
H. influenzae showed sensitivity to chloramphenicol and ceftriaxone as well as moderate resistance to tetracycline. This however, differs from findings of a similar study done in Namibia, where H. influenzae resistance to chloramphenicol, ceftriaxone and tetracycline was noted [
Group B streptococcus was recorded with the findings of 100% resistance to tetracycline and 100% sensitivity to penicillin and erythromycin as shown in
Although bacterial culture is regarded as a gold standard in the diagnosis of meningitis, the use of antibiotics prior to collection of CSF might attribute to false negative culture results, which therefore necessitates the use of non-culture techniques such as PCR for diagnosis. PCR is the most accurate and reliable method among the non-culture techniques and it is a highly sensitive and specific method which detects specific bacterial DNA in CSF [
For the comparison of culture and PCR in this study, the findings of this current study showed that in some culture negative cases, PCR was positive, as shown in
Studies have shown that Gram stain identifies meningitis causing bacteria in 60% - 90% of patients with suspected meningitis [
Gram stain yields positive results in 10% - 15% of patients with bacterial meningitis, who test negative with culture results [
In this study, Gram stain was positive in 67.8% of all culture positive cases as shown in
Bacterial meningitis leads to a predominance of neutrophils in CSF in about 80% - 95% of bacterial meningitis cases [
Furthermore, our study found that 1.4% of samples with bacterial growth had a neutrophil count less than 5/mm3, which does not correlate with indications of bacterial meningitis. Falsely decreased neutrophil count in meningitis can be attributed to the lysis of neutrophils in the CSF sample due to delay in cell count [
Our findings indicated that 78.8% of samples with elevated neutrophil count (>5/mm3) had no bacterial growth. In this case, elevated CSF neutrophil count signifies the presence of bacteria in CSF, however as there was no bacterial growth detected; this might be due to the loss of bacterial viability due to unsuitable transport and storage conditions or excessive manipulation of the CSF sample. Also, WBCs may be present in CSF due to intracranial haemorrhage.
Elevated CSF protein is an indicator of bacterial meningitis. Our study findings showed that only 25.4% of samples with elevated protein were culture positive, as shown in
Laboratory findings of reduced CSF glucose in relation to plasma glucose signify the presence of bacteria in CSF, which metabolises CSF glucose [
We found out that 18.9% of CSF samples that had low glucose level reported bacterial growth by culture, as shown in
In the current study, we concluded that the most frequent meningitis-causing bacterium isolated from CSF of children below five years in Windhoek was S. pneumoniae. S. pneumoniae was highly resistant to penicillin and cotrimoxazole, and sensitive to ciprofloxacin, vancomycin and ceftriaxone. Although isolated in one case only, group B streptococcus was sensitive to penicillin and resistant to tetracycline.
There was a statistically significant association between CSF culture results and PCR results in detecting meningitis-causing bacteria in CSF. There was also statistically significant association between culture results and Gram stain results.
Erastus Lafimana Haimbodi,Munyaradzi Mukesi,Sylvester Rodgers Moyo,Owen Mtambo, (2016) Frequency of Bacterial Organisms Isolated from Cerebrospinal Fluid (CSF) of Children under Five Years in Windhoek from 2010 to 2014. Open Journal of Medical Microbiology,06,125-132. doi: 10.4236/ojmm.2016.63017