Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Increased creatine kinase brain isoenzyme concentration in cerebrospinal fluid with meningitis

CPK-BB (CK-BB) isoenzyme is an intracellular enzyme released in various neurologic conditions, including central nervous system (CNS) infections. Activity of CK-BB in cerebrospinal fluid (CSF) was determined in 80 children by electrophoresis and densitometry. The possible correlation between CNS infection and CK concentrations was assessed. Significantly elevated concentrations of CK activity (P < 0.01) in the CSF were found in children with bacterial meningitis as compared with children with either aseptic meningitis or normal CSF findings. The data suggest the possibility of utilizing CSF CK activity to differentiate between bacterial and viral meningitis in situations where a routine CSF examination is inconclusive.     

Meningitis caused by Streptococcus bovis

Streptococcus bovis was isolated from the CSF of a 66-year-old man with meningitis. His clinical appearance was unusual in that he lacked typical signs and symptoms of pyogenic meningitis. Streptococcus bovis was also recovered from his blood, which suggested that bacterial endocarditis was the source of his CNS infection. He was cured after four weeks of therapy with intravenous penicillin G potassium. This is the fourth reported case of meningitis caused by S bovis. The previous three patients also had endocarditis caused by S bovis. Because of the reported propensity of S bovis to infect heart valves and the frequent association of S bovis bacteremia with malignant gastrointestinal (GI) tract tumors, recovery of this organism form CSF should prompt a search for bacterial endocarditis and occult GI cancer.     

Photometric application of the Gram stain method to characterize natural bacterial populations in aquatic environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.     

Central nervous system infection with Listeria monocytogenes. 33 years' experience at a general hospital and review of 776 episodes from the literature

We reviewed 776 previously reported and 44 new cases of CNS listeriosis outside of pregnancy and the neonatal period, and evaluated the epidemiologic, diagnostic, and therapeutic characteristics of this infection. Among patients with Listeria meningitis/meningoencephalitis, hematologic malignancy and kidney transplantation were the leading predisposing factors, but 36% of patients had no underlying diseases recognized. The infection occurred throughout life, with a higher incidence before the age of 3 and after the age of 45-50 years. Fever, altered sensorium, and headache were the most common symptoms, but 42% of patients had no meningeal signs on admission. Compared with patients with acute meningitis due to other bacterial pathogens, patients with Listeria infection had a significantly lower incidence of meningeal signs, and the CSF profile was significantly less likely to have a high WBC count or a high protein concentration. Gram stain of CSF was negative in two-thirds of cases of CNS listeriosis. One-third of patients had focal neurologic findings, and approximately one-fourth developed seizures over their course. Mortality was 26% overall, and was higher among patients with seizures and those older than 65 years of age. Relapse occurred in 7% of episodes. Ampicillin for a minimum of 15-21 days (with an aminoglycoside for at least the first 7-10 days) remains the treatment of choice. Cerebritis/abscess due to L. monocytogenes, without meningeal involvement, is less common but may be diagnosed by blood cultures and CNS imaging, or by stereotactic biopsy. Longer antibiotic therapy (at least 5-6 weeks) is needed in the presence of localized CNS involvement.     

Lysozyme activity in cerebrospinal fluid. Studies in inflammatory and non-inflammatory CNS disorders

Lysozyme activity was measured in cerebrospinal fluid (CSF) from 114 patients with inflammatory (bacterial and serous meningitis, polyradiculitis, encephalitis) and non-inflammatory (multiple sclerosis, CNS tumors, cerebral vascular diseases) CNS diseases. Highly elevated values were found consistently in patients with bacterial meningitis. Elevated values were found also in patients with encephalitis, polyradiculitis, multiple sclerosis and CNS tumors, but a considerable overlapping between these groups and normal controls precludes the use of CSF lysozyme measurements as a diagnostic aid in the latter disease groups. Simultaneous measurements of lysozyme, albumin and IgG in CSF and serum suggested that the mechanism for increased CSF lysozyme values in bacterial meningitis is mainly a breakdown of the blood/brain barrier, whereas the increased CSF lysozyme values in the remaining groups of patients are more likely caused by production of lysozyme by cells within the meninges (neutrophilic granulocytes, monocytes?).     

Gram stain method shows better sensitivity than clinical criteria for detection of bacterial vaginosis in surveillance of pregnant, low-income women in a clinical setting

OBJECTIVE: The purpose of the study is to determine whether the Gram stain method is superior to the clinical criteria for the diagnosis of bacterial vaginosis in low-income pregnant women seen in a resident clinic setting. The clinical criteria is the current diagnostic method employed to diagnose bacterial vaginosis. STUDY DESIGN: In this study, 51 pregnant women with vaginal discharge were prospectively evaluated. All were screened using the clinical criteria, Gram stain method, and culture of the discharge. The modified scoring system instituted by Nugent et al. (J Clin Microbiol 29:297-301, 1991) was employed in reading the Gram stain smears. The clinical criteria were then compared with the Gram stain method. Isolation of moderate to many Gardnerella vaginalis growth by culture was used as the confirmatory finding. RESULTS: Sensitivity of the Gram stain method (91%) was significantly higher than that of the clinical criteria (46%), (sign test P = 0.0023, < 0.01). The Gram stain method also has both a low false-negative (4%) and high negative predictive value (96%), making it an ideal diagnostic test. CONCLUSION: The Gram stain method is a rapid and cost-effective test that is also highly reproducible and readily available in many laboratories. These features make the Gram stain method a more desirable screening procedure for bacterial vaginosis in a clinic population.     

No high affinity melatonin binding sites are detected in murine melanoma cells and in normal human melanocytes cultured in vitro

Samples of 1815 cerebrospinal fluid (CSF) were studied in a meningitis outbreak during 1989 in S?o Paulo, Brazil. Neisseria meningitis 56% with 44% type B, Haemophilus influenzae 17%, from which 72% in children (days to 3-year-old) and Streptococcus pneumoniae 14% from which 60% in children (day to 1-year-old) of 443 (24%) of all strains. Cytochemistry study showed: purulent or turbidity aspects in 70 to 79% positive bacterioscopy or culture of CSF; white cells count > 500/mm3; glucose < 45 mg/dl; protein > 90 mg/dl in 90% of all patients. We concluded that: CSF prognostic factors: (aspect and cytochemistry) were correlated with bacterial meningitis. Bacterioscopy and positive cultures were correlated to NM, SP and HI isolation from these patients (Goodman Test).     

Group B meningococcal meningitis in India

The first case of infection with Group B meningococcus in India is reported. The patient was a 4-month-old boy who presented with meningitis and died within 6 h of admission. Gram stain of CSF showed meningococci and latex particle agglutination test on CSF was strongly positive for Neisseria meningitidis serogroup B. The CSF was also positive for meningococcus by polymerase chain reaction using primers NM1 and NM6, which amplify a 650 bp region of the dihydropteroate synthase (dhps) gene of N. meningitidis.     

Basic fibroblast growth factor in experimental and clinical bacterial meningitis

Basic fibroblast growth factor (bFGF), a neurotrophic factor in the CNS, is expressed at high levels in response to seizures or strokes. We examined the expression of bFGF during experimental bacterial meningitis and the levels of bFGF in the cerebrospinal fluid (CSF) of children with bacterial meningitis. For the experimental study, a mouse model of meningitis was established by intracranial injection of Streptococcus pneumoniae. Twenty-four hours after induced meningitis, the brains were sectioned and stained immunohistochemically for bFGF. Neutrophils and macrophages infiltrating the leptomeninges and the ventricles exhibited strong bFGF immunoreactivity. The neurons in the areas adjacent to the inflamed ventricles also showed enhanced bFGF expression. For the clinical study, we used an enzyme immunoassay to measure bFGF in CSF in 18 children with bacterial meningitis, 12 with aseptic meningitis, and 18 controls. The CSF levels of bFGF were twice as high in children with bacterial meningitis (medians 6.75-7.21 pg/mL) compared with those who had aseptic meningitis (2.9 pg/mL) or in control subjects (2.65 pg/mL, p < 0.0001, respectively). In patients with bacterial meningitis who survived, CSF bFGF decreased significantly after 24-50 h of antibiotic therapy (p < 0.0005). Patients who developed major sequelae or died had much higher levels of CSF bFGF than those without (134.9 pg/mL versus 7.38 pg/mL, p < 0.05). These findings of enhanced immunoreactivity of bFGF in experimental bacterial meningitis and an association of CSF levels of bFGF with disease severity in childhood bacterial meningitis suggest a biologic role for this neurotrophic factor in the pathophysiology of bacterial meningitis.     

The value of amniotic fluid interleukin-6, white blood cell count, and gram stain in the diagnosis of microbial invasion of the amniotic cavity in patients at term

PROBLEM: Subclinical microbial invasion of the amniotic cavity occurs in 18.8% of women with term labor and intact membranes and in 34% of patients with term PROM and is a risk factor for the development of puerperal infection related morbidity. Although amniotic fluid white blood cell count, interleukin-6 determination, and Gram stain examination have been used for the diagnosis of intrauterine infection in patients with preterm labor and preterm premature rupture of membranes, no information is available about the accuracy and specific cut-off values for these tests in patients at term. The purpose of this study was to compare the performance of the amniotic fluid Gram stain examination, white blood cell count, and interleukin-6 determination in the identification of microbial invasion of the amniotic cavity in patients at term with and without PROM. METHOD: Amniotic fluid was retrieved from 148 patients with term gestations (90 patients with spontaneous labor and intact membranes and 58 patients with PROM). Samples were cultured for bacteria and Mycoplasma species. Amniotic fluid Gram stain, white blood cell count, and interleukin-6 determinations (ELISA, sensitivity: 43 pg/ml) were performed in all samples. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture for microorganisms. Analysis was conducted using Mann-Whitney U test, Fisher's exact test, receiver operating characteristic curves and logistic regression. RESULTS: Patients with spontaneous labor and intact membranes: The prevalence of microbial invasion of amniotic cavity in this group was 15.6% (14/90). The most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determination (sensitivity for: interleukin-6 > or = 5.7 ng/ml = 86%, white blood cell count > or = 20 cells/mm3 = 64%, Gram stain = 28%). The most specific test was the Gram stain of the amniotic fluid (specificity for: Gram stain = 84%, interleukin-6 = 79% and white blood cell count = 63%). Multiple logistic regression demonstrated that amniotic fluid interleukin-6 concentration was the only covariate that retained statistical significance when intrauterine infection was used as outcome variable. Patients with PROM: The prevalence of a positive amniotic fluid culture in this group was 39.7% (23/58). Logistic regression demonstrated that only interleukin-6 retained a significant relationship with the results of amniotic culture when all variables were entered simultaneously into a model to predict amniotic fluid culture results. The most sensitive tests for the detection of intrauterine infection were interleukin-6 determination and white blood cell count (sensitivity for interleukin-6 > or = 3.4 ng/ml and white blood cell count > or = 20 cells/mm3 = 69.6% for both). The most specific test was Gram stain (97.1%). CONCLUSIONS: Amniotic fluid interleukin-6 determination is the best rapid test for the detection of microbial invasion of the amniotic cavity in patients at term with and without PROM. When this test is not available, amniotic fluid Gram stain and white blood cell count represent valid diagnostic tools to assess the microbial state of amniotic cavity.     

Evaluation of Calcofluor staining in the diagnosis of fungal corneal ulcer

For laboratory diagnosis of mycotic keratitis, demonstration of fungal pathogens on direct microscopy and their isolation by culture is essential. The addition of Calcofluor white (CFW) stain to the diagnostic armamentarium has significantly increased the sensitivity of smear examination on direct microscopy. During a period of 1 year, 143 consecutive patients with corneal ulcers were investigated by direct microscopy with potassium hydroxide (KOH) wet mount, Calcofluor white stain and routine cultures of corneal scrapings. Fungi were detected as aetiological agents in 21 (15%) patients. Different species of the genera Aspergillus (35%), Fusarium (23%), Acremonium (12%), Paecilomyces (12%), Cladosporium (6%), Alternaria (6%) and Pseudallescheria (6%) were the common isolates. Calcofluor white stain on direct microscopy detected fungi in 20 (95.2%) patients in comparison with 15 (71.4%) patients by both KOH wet mount examination and culture. Calcofluor white stain was significantly more sensitive than KOH wet mount in demonstrating fungal pathogens.     

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis. The European Union Concerted Action on Viral Meningitis and Encephalitis

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the "gold standard" were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.     

Ventriculoperitoneal shunt infections with gram-negative bacteria

Infection causes major morbidity and mortality in patients with cerebrospinal fluid (CSF) shunts. The prognosis of CSF shunt infections caused by Gram-negative bacteria (GNB) has been thought to be particularly poor. The authors reviewed all GNB shunt infections treated at Children's Memorial Hospital from January 1986 to January 1990 (n = 23). Of these infections 20 (87%) occurred within 4 weeks after shunt revision (median, 10 days). The most frequent symptoms were fever, lethargy, and irritability; the illness was not severe in the majority of these patients. Escherichia coli was isolated from 12 of 23 patients (52%), Klebsiella pneumoniae from 5 (22%), and mixed GNB from 3 (13%) patients. Initial treatment always included immediate shunt removal, externalized ventricular drainage, and intravenous antibiotics. Extraventricular drainage revision and/or intraventricular antibiotics were required in four patients whose CSF cultures were persistently positive for GNB. At admission, these patients had CSF glucose levels of < 10 mg/dl and CSF positive for GNB by Gram's stain. The overall cure rate was 100%, and no recurrence was observed; however, a subsequent infection with a different organism developed in four patients. Only 2 of 19 patients (11%) who were followed up suffered apparent CNS damage. One patient died of unrelated causes shortly after treatment. Our findings indicate that 1) patients with GNB CSF shunt infections often appear relatively well at presentation; 2) CSF positive for GNB by Gram's stain and very low CSF glucose levels predict continued positive CSF cultures, despite appropriate antibiotic therapy; and 3) GNB CSF shunt infections can be successfully treated by prompt shunt removal, extraventricular drainage, and intravenous antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)     

The value of C-reactive protein for the differentiation of bacterial meningitis from viral meningitis

In order to differentiate bacterial meningitis versus viral meningitis, we have comparatively tested the efficacy of the following tests: C-reactive protein (CRP), erythrocytes sedimentation rate (ESR), fever, level of glucose in cerebro-spinal fluid (CSF), glucose in CSF/glycemia ratio, number of white blood cells in peripheric blood, percentage of neutrophils in peripheric blood, level of proteins in CSF and number of nucleated cells in CSF for a group of 49 patients, both children and adults with central nervous system infection (37 patients with bacterial meningitis and 12 with viral meningitis) hospitalised between May 1993 and July 1994 in Clinical Hospital for Infectious Diseases in Ia?i. The mean value of CRP in bacterial meningitis patients was 8.78 mg%, contrasting with the mean value of CRP = 1.92 mg% recorded in patients with viral meningitis. Ten out of 37 bacterial meningitis patients presented a CRP concentration < 1.85 mg%. All these 10 patients have already had an antibiotic treatment at the moment of the assay. One out of 12 cases of viral meningitis had a value of CRP = 3.3 mg%, all the remainder cases having values under 1.85 mg%. We recorded highly significant differences between the two patient groups for CRP (p < 0.001), ESR (p < 0.01), protein concentration in CSF (p < 0.001) and number of nucleated cells in CSF (p < 0.001). Differences recorded for fever, concentration of glucose in CSF, glucose in CSF/glycemia ratio, number of leucocytes in peripheric blood and percentage of neutrophils in peripheric blood, were not significant (p > 0.5). Data were analysed also by box-plot method which facilitates the visual appraisal of the differences recorded between the two aetiological groups. In conclusion, assays of CRP and ESR may be used as differentiation tests for bacterial meningitis versus viral meningitis, when assay is done before the antibiotic treatment, being sufficient sensitive, and easy to perform.     

Moraxella (Branhamella) catarrhalis: its isolation in the respiratory secretions of adult patients

OBJECTIVE: We have designed a retrospective study in order to know the clinical significance of the isolation of Moraxella (Branhamella) catarrhalis (MC) in respiratory specimens of adult hospitalized patients. METHODS: We performed a Gram stain and culture on blood-agar, MacConkey media and quantitative culture in chocolate-agar to all respiratory samples. In patients with a clinical diagnosis of pneumonia BCYE-alpha was added. During 2 years (1992-1993) MC was isolated in respiratory specimens from 52 patients. We revised the clinical history of all these patients. RESULTS: MC was isolated in 60 respiratory specimens (sputum and/or tracheobronchial aspirates) from 52 patients. The Gram stain showed gram-negative cocci in 77% and gram-positive cocci in 17% of the cases. MC grew in pure culture in 28 specimens (46.6%). In 23% of cases MC was isolated with Streptococcus pneumoniae and in 21% with Haemophilus influenzae. Fifty-two stocks (86.6%) produced beta-lactamase. Twelve patients had a clinical diagnosis of pneumonia, 8 of them had an underlying chronic respiratory disease. Other 24 patients with an underlying chronic respiratory disease had a bronchial infection as a cause of exacerbation of their respiratory disease. Seven patients without an underlying chronic respiratory disease had a clinical episode of acute bronchitis. Finally, in 9 patients the isolation of MC was considered a colonization. CONCLUSIONS: In 17% cases MC was identified as a gram-positive cocci in the Gram stain, which may cause false diagnosis. The etiological importance of MC in episodes of acute exacerbation of patients with an underlying chronic respiratory disease is high.     

Bacteroides fragilis meningitis revealing a meningorectal fistula

Cases of meningitis due to Bacteroides fragilis are rare; we report a case revealing a meningorectal fistula. CASE REPORT: A 2-month-old infant developed a severe sepsis syndrome following a rectosigmoidoscopy for rectal bleeding. Lumbar puncture diagnosed bacterial meningitis. Cerebrospinal fluid (CSF) culture evidenced B fragilis with betalactamase. The initial antibiotherapy was changed for imipenem-metronidazole, which is at present the recommended antibiotherapy. Malformation including pre-spinal tumor and meningorectal fistula was evoked on magnetic resonance imaging (MRI) and confirmed by surgery. The outcome was favorable after surgery and antibiotherapy. CONCLUSION: B fragilis meningitis are usually associated with sepsis, whose origin is obvious. In our case, meningitis was isolated, revealing a meningorectal fistula.     

A clinical, microbiological, and histopathologic study of trichostasis spinulosa

Trichostasis spinulosa (TSS) is a relatively common follicular disorder that can occur on the face and trunk, especially in the interscapular area. Its cause remains unclear. We examined clinically 30 patients with TSS and follicular materials extracted from each patient were examined microscopically. Bacterial culture and skin biopsy were done in 12 and 10 patients, respectively. Periodic acid Schiff (PAS) and Brown-Brenn Gram stain were used for detection of pityrosporum (malassezia) and bacteria. The interscapular area (14/30), nose (8/30), and cheek (4/30) were common sites of TSS. Pityrosporum and bacteria in the extracted follicular material were found at the rates of 82.6% and 73.3%, respectively. In histologic examination, follicular hyperkeratosis and numerous vellus hairs enveloped within keratotic sheath were common features. Pityrosporum and bacteria were found at the rate of 70% in biopsied specimens on PAS and Brown-Brenn Gram stain. In bacterial culture, Propionibacterium acne was most commonly identified in 75% (9 out of 12 patients). Pityrosporum and bacteria, especially Propionibacterium acne, were commonly found in the extracted follicular material and biopsied specimens. Thus, they may be related to the induction of follicular hyperkeratosis with retention of vellus hairs, and we suggest that these microorganisms may be one of the possible etiologic factors of TSS.     

Experimental reproduction of a spiking mortality syndrome of turkeys

We measured the levels of interleukin-6 (IL-6), albumin, C-reactive protein (CRP) and alpha 2 macroglobulin (alpha 2M), all of which have different spectrums of molecular weight, in the cerebrospinal fluid (CSF) and serum in 121 patients to evaluate damage to the blood-cerebrospinal fluid barrier (BCB) in meningitis. There was an extraordinary high level of IL-6 in the CSF when patients had bacterial or viral meningitis, but the level returned to a normal range within a week in almost all of these cases. There were no significant differences in CSF albumin levels among the different disease groups. The CRP level in CSF is considered to correlate with the serum level, and CSF CRP was higher in bacterial meningitis than in viral meningitis, however, CRP in CSF was increased in some of the infectious diseases without meningitis. The alpha 2M in CSF, which tends to be at extraordinarily high levels when there is damage to the BCB, correlated highly with CSF cell counts. CSF IL-6 seemed to be a useful indicator to identify the acute active phase of meningitis. CRP and alpha 2M in CSF are considered to be useful to differentiate bacterial meningitis, bacterial infection without meningitis and viral meningitis. Extraordinarily high levels of alpha 2M, which has a high molecular weight, in CSF is indicative of BCB damage.