Normocellular Community-Acquired Bacterial Meningitis in Adults: A Nationwide Population-Based Case Series.

STUDY OBJECTIVE: This study sought to describe the clinical presentation of normocellular community-acquired bacterial meningitis in adults. METHODS: Using the prospective, nationwide, population-based database of the Danish Study Group of Infections of the Brain, the study identified all adults with normocellular community-acquired bacterial meningitis who were treated at departments of infectious diseases in Denmark from 2015 through 2018. Normocellular community-acquired bacterial meningitis was defined as a cerebrospinal fluid leukocyte count of up to 10×106/L combined with detection of bacteria in the cerebrospinal fluid. Outcome was categorized according to the Glasgow Outcome Scale at discharge. RESULTS: Normocellular cerebrospinal fluid was observed in 12 of 696 (2%) patients with community-acquired bacterial meningitis. The median age was 70 years (range 17 to 92 years), and 8 of 12 (67%) patients were male. All patients had symptoms suggestive of community-acquired bacterial meningitis and pathogens identified by culture (Streptococcus pneumoniae, n=10; Staphylococcus aureus, n=1) or polymerase chain reaction (Neisseria meningitidis; n=1) of the cerebrospinal fluid. Bacteremia was found in 9 of 12 (75%) patients, and 1 of 12 (8%) presented with septic shock. None of the patients had serious underlying immunocompromising conditions. The median times from admission to lumbar puncture and meningitis treatment were 2.5 hours (interquartile range 1.1 to 3.9 hours) and 2.6 hours (interquartile range 0.9 to 22.8 hours). In 3 of 11 (27%) patients, empiric treatment for community-acquired bacterial meningitis was interrupted by a normal cerebrospinal fluid cell count. The overall case-fatality rate was 3 of 12 (25%); meningitis treatment was interrupted in 1 of these patients, and 8 of 12 (67%) had a Glasgow Outcome Scale score of 1 to 4 at discharge. CONCLUSION: Normocellular community-acquired bacterial meningitis is not very common, but it is important to consider and may be associated with a pneumococcal cause.

[Meningococcemia: Different Serotypes in the Same Region]. / Meningokoksemi: Ayni Cografyada Farkli Serotipler.

Meningococcal infections are important health problems causing high morbidity and mortality. Neisseria meningitidis have 13 serogroups. A, B, C, Y and W135 are the most common causes of invasive disease among those serogroups. The distribution of the serogroups differs according to the geographical regions and the age groups. In this case report, two cases of meningococcemia infected with serogroup C and Y of N.meningitidis rarely seen in our country were presented. First case was a two and a half year-old female patient who has admitted to our pediatric emergency unit with fever and rash spreading from lower extremities to her body. The patient had diffuse purpuric rash with generalized weakness and tendency to sleep at admission. The patient has been suspected as meningococcemia because of the skin rash, tendency to sleep and hypotension. Antibiotics treatment was started immediately and lumber puncture was performed. In blood tests, leukocyte count: 3600/mm3 (61% neutrophils), hemoglobin: 11.1 g/ dl, platelet count: 127.000/mm3 , C-reactive protein: 10 mg/dl, erythrocyte sedimentation rate: 6 mm/ hour, prothrombin time: 28.8 seconds (normal value= 11-16), prothrombin activity: 36%, international normalized ratio (INR): 2.13 (normal value= 1-1.5), activated partial thromboplastin time: 57.7 seconds (normal value= 25-35 sec), fibrinogen: 246 mg/dl (normal value= 200-400 mg/dl) and in cerebrospinal fluid protein: 21 mg/dl and glucose: 62 mg/dl were found. There were eight cells in the microscopic examination. Skin rashes were increased and the patient became hypotensive. No microorganisms were isolated in blood and cerebrospinal cultures. N.meningitidis serogroup C was isolated from the cerebrospinal fluid of the patient using polymerase chain reaction (PCR). The patient suffered from immune-mediated arthritis in the sixth day of treatment and nonsteroidal anti-inflammatory drugs were given. The patient has recovered with antibiotics, fresh frozen plasma and inotropic treatment. Second case was a 13 year-old male patient who has admitted three days after the first case with a pre-diagnosis of malignancy because of pancytopenia and fever. The patient had generalized weakness and a few petechial purpuric rashes at the facial region at admission. After the admission general status of the patient has worsened rapidly and he has died as a result of cardiovascular arrest. Blood tests in admission showed leukocyte count: 6000/mm3 (79% neutrophils), hemoglobin: 17.3 mg/dl, platelet count: 16.000/mm3 , C-reactive protein: 8.63 mg/dl, prothrombin time: 92.6 seconds, prothrombin activity: 10%, INR: 6.78, activated partial thromboplastin time: 231.5 seconds. Cerebrospinal fluid obtained from postmortem lumbar puncture showed no growth (protein: 95 mg/dl, glucose: 35 mg/dl) and N.meningitidis serogroup Y was detected by PCR. Two meningococcemia cases caused by two different serogroups which are rarely seen in our region in recent years were presented at the same time period in the same hospital. This case report pointed out that surveillance has a great importance in such diseases.

Molecular interactions between Neisseria meningitidis and its human host.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonises the nasopharynx of humans. For an unknown reason, N. meningitidis can cross the nasopharyngeal barrier and invade the bloodstream where it becomes one of the most harmful extracellular bacterial pathogen. This infectious cycle involves the colonisation of two different environments. (a) In the nasopharynx, N. meningitidis grow on the top of mucus-producing epithelial cells surrounded by a complex microbiota. To survive and grow in this challenging environment, the meningococcus expresses specific virulence factors such as polymorphic toxins and MDAΦ. (b) Meningococci have the ability to survive in the extra cellular fluids including blood and cerebrospinal fluid. The interaction of N. meningitidis with human endothelial cells leads to the formation of typical microcolonies that extend overtime and promote vascular injury, disseminated intravascular coagulation, and acute inflammation. In this review, we will focus on the interplay between N. meningitidis and these two different niches at the cellular and molecular level and discuss the use of inhibitors of piliation as a potent therapeutic approach.

Standing on the shoulders of giants: two centuries of struggle against meningococcal disease.

Meningococcal disease was first clinically characterised by Gaspard Vieusseux in 1805, and its causative agent was identified by Anton Weichselbaum in 1887, who named it Diplococcus intracellularis menigitidis. From the beginning, the disease was dreaded because of its epidemic nature, predilection for previously healthy children and adolescents, and high mortality. In the last decade of the 19th century, the concept of serum therapy for toxin-related bacterial diseases was identified. This concept was applied to meningococcal disease therapy, in an independent way, by Wilhelm Kolle, August von Wasserman, and Georg Jochmann in Germany, and Simon Flexner in the USA, resulting in the first successful approach for the treatment of meningococcal disease. During the first three decades of the 20th century, serum therapy was the standard treatment for meningococcal disease. With the advent of sulphamides first and then antibiotics, serum therapy was abandoned. The great challenges that infectious diseases medicine is facing and the awaiting menaces in the future in terms of increasing antibiotic resistance, emergence of new pathogens, and re-emergence of old ones without effective therapy, make passive immunotherapy a promising tool. Acknowledging the achievements of our predecessors might teach us some lessons to bring light to our future.

Value of the eazyplex® CSF direct assay in rapid diagnosis of invasive meningococcal disease - Case report.

There is a need for easy-to-use molecular assays for diagnosis of invasive meningococcal disease. Here, we report the rapid identification of Neisseria meningitidis in a cerebrospinal fluid sample from a patient with purulent meningitis using a commercially available loop-mediated isothermal amplification assay, resulting in a prompt de-escalation of the initial empiric antibiotic therapy.

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage.

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

Diagnostic accuracy of loop-mediated isothermal amplification as a near-patient test for meningococcal disease in children: an observational cohort study.

BACKGROUND: Diagnosis of meningococcal disease relies on recognition of clinical signs and symptoms that are notoriously non-specific, variable, and often absent in the early stages of the disease. Loop-mediated isothermal amplification (LAMP) has previously been shown to be fast and effective for the molecular detection of meningococcal DNA in clinical specimens. We aimed to assess the diagnostic accuracy of meningococcal LAMP as a near-patient test in the emergency department. METHODS: For this observational cohort study of diagnostic accuracy, children aged 0-13 years presenting to the emergency department of the Royal Belfast Hospital for Sick Children (Belfast, UK) with suspected meningococcal disease were eligible for inclusion. Patients underwent a standard meningococcal pack of investigations testing for meningococcal disease. Respiratory (nasopharyngeal swab) and blood specimens were collected from patients and tested with near-patient meningococcal LAMP and the results were compared with those obtained by reference laboratory tests (culture and PCR of blood and cerebrospinal fluid). FINDINGS: Between Nov 1, 2009, and Jan 31, 2012, 161 eligible children presenting at the hospital underwent the meningococcal pack of investigations and were tested for meningococcal disease, of whom 148 consented and were enrolled in the study. Combined testing of respiratory and blood specimens with use of LAMP was accurate (sensitivity 89% [95% CI 72-96], specificity 100% [97-100], positive predictive value 100% [85-100]; negative predictive value 98% [93-99]) and diagnostically useful (positive likelihood ratio 213 [95% CI 13-infinity] and negative likelihood ratio 0·11 [0·04-0·32]). The median time required for near-patient testing from sample to result was 1 h 26 min (IQR 1 h 20 min-1 h 32 min). INTERPRETATION: Meningococcal LAMP is straightforward enough for use in any hospital with basic laboratory facilities, and near-patient testing with this method is both feasible and effective. By contrast with existing UK National Institute of Health and Care Excellence guidelines, we showed that molecular testing of non-invasive respiratory specimens from children is diagnostically accurate and clinically useful. FUNDING: Health and Social Care Research and Development, Public Health Agency, Northern Ireland.

nspA gene as a specific genetic marker for detection of Neisseria meningitidis causing bacterial meningitis.

Bacterial meningitis caused by Neisseria meningitidis which causes human brain meninges damage, is generally diagnosed from patient cerebrospinal fluid through microscopy, immunological assays, biochemical test, PCR, microarray and biosensors. However, these methods are expensive, time-consuming or non-confirmatory due to certain limitations. A quick PCR based method was developed for detection of bacterial meningitis caused by N. meningitidis using specific primers based on amplification of virulence nspA (Neisseria surface protein A) gene partial sequence (202 bp). The nspA gene amplicon could be used as a genetic marker for minimum detection of 10 ng genomic DNA (G-DNA) of N. meningitidis with high sensitivity only in 80 min, which is least time reported for the confirmation of the disease. However, the lower detection limit was found as low as 1.0 ng G-DNA, but with less sensitivity. The cross-reactivity of the genetic marker, was also studied with other possible pathogens. A comparison with the presently available detection methods and our method was also done using patient samples.

Influence of antibiotic therapy prior to admission on the efficacy of classical methods for the diagnosis of meningococcal disease.

AIM: To assess the influence of preadmission antibiotic therapy on the results of the classical methods for bacteriological confirmation of meningococcal disease (MD). MATERIAL AND METHODS: Retrospective study of the MD cases diagnosed in the "St. Parascheva" Universitary Clinical Infectious Diseases Iasi between 1994 and 2011. RESULTS: The etiological diagnosis was made by identifying the meningococcus in the CSF (cerebrospinal fluid) in 71.9% of the 323 patients and by blood culture in 8%. Preadmission antibiotic therapy received 39% of the patients, thus the sensitivity of test was significantly reduced: direct examination from 64.6% to 43.2% (p < 0.001), cultures from 55.9% to 27.2% (p < 0.001), and latex-agglutination from 84.6% to 58.8% (p = 0.003). The rate of positive CSF decreased from 82.1% to 56% (p < 0.001). Preadmission antibiotic therapy significantly increased the ratio of cases in which meningococcus was not detected in CSF by any of the classical methods (44% compared to 17.9% in the cases without prior treatment). The proportion of cases in which meningococcal isolation was done by two methods decreased from 38.5% to 19.2%, and of those by all three methods from 16.9% to 5.6% (p < 0.001). Preadmission antibiotic therapy also decreased the rate of positive blood cultures from 14.7% to 3.5% (Fisher's exact test, p = 0.009). CONCLUSIONS: Antibiotic treatment prior to admission significantly decreases the percentage of patients with MD in which meningococcal isolation can be done; this requires the use of a more sensitive diagnosis method (ex. qPCR).

[Epidemiology of meningococcal infection in Georgia].

Epidemiology of meningococcal infection in Georgia was studied for 2009-2011. As the official surveillance data shows, morbidity with the infection is characterized by a downward trend. During the analyzed period the morbidity level declined by 2,4 times. The average incidence rate for the period composed 0,95 ± 0,12 per 100 000 population. The infection is unequally spread throughout the country. High incidence is indicated in Tbilisi city and the regions: Ajara and Kvemo Kartli. Children are the most vulnerable population. 81,3% of all the cases fell on the children' population. High incidence rate in children of the age under 1 year is especially alarming, composing 11,52 per 100 000 population. Irrespective of the downward trend meningococcal infection in Georgia remains as a severe disease, high level of lethal outcome (lethality 18,6±3,0%) is the confirmation of that. Lethality is even higher in small children under 1 year - 27,7 ± 7,4%. The study of the isolated N. meningitides from patients with meningococcal meningitides as well as with meningococcemia showed that N. meningitides of the serosgoups B and C are being circulated in Georgia. Epidemiological investigation of the infection foci in terms of medical examination of contact persons of the cases indicated that the causative agents of the same serogroups are also widely circulated among contact subjects. Along of the passive surveillance data on the meningococcal disease, results of active investigation of epidemiological foci of the infection may serve as valuable information for planning of specific prevention measures against the infection.

Characterization of Neisseria meningitidis isolates from Egypt using multilocus sequence typing.

To characterize Neisseria meningitidis isolates collected from cerebrospinal fluid of meningitis cases in Egypt (1998-2003) as part of surveillance studies, 67 isolates were serogrouped, tested for antibiotic sensitivity and analyzed using multilocus sequence typing (MLST). Results show that isolates expressing serogroup B (50.7%) and serogroup A (34.3%) antigens were predominant in Egypt during the surveillance period, possibly due to suppression of other serogroups by meningococcal vaccines in current use. Intermediate resistance to penicillin was observed in 71% of the isolates, suggesting a need for physicians to shift to third-generation cephalosporins during the empirical treatment of infection. Recurrent lineages of N. meningitidis in Egypt appear to originate from Europe and other Middle Eastern countries. Of 19 sequence types detected, five were unique to Africa and 10 were not observed previously in the MLST database. The information obtained illustrates the changing dynamics of meningitis after vaccine introduction in Egypt.

Diagnosis of meningococcal infection by QPCR: detection and quantification of DNA / Neisseria meningitidis.

We present the case of a patient with clinical signs of meningococcemia and negative bacterial culture. Microbiological diagnosis was possible only by testing with real time PCR technique the cerebrospinal fluid and blood samples. Both pathologic samples were positive for Neisseria meningitidis by this rapid and sensitive diagnostic method.

The in vitro susceptibility to 7 antibiotics of Neisseria meningitidis strains isolated last years in Romania.

Thirty-one strains of Neisseria meningitidis strains coming from CSF, collected at the National Reference Center for Meningococci between January 2007-March 2008 were serogrouped and analysed for the susceptibility to 7 antibiotics: penicillin (Pc), erythromycin (Em), ceftriaxone (Cro), cefotaxime (Ctx), ciprofloxacin (Cip), rifampin (Rif), sulfonamides (Sm). Results revealed that serogroup B accounted to the highest number of strains (51.7%), followed by serogroup C (35.5%), serogroup W 135 (6.4%) and serogroup X (6.4%). The data analysed according to EMGM revealed the following aspects: 100% susceptibility to Cro and Ctx., 96.7% susceptibility to Rif (MIC 50 and MIC 90 =0.03 mg/l) and 96.7% susceptibility to Cip (MIC 50 and MIC 90=0.0015 mg/l). Concerning Pc, it was revealed 58% susceptible strains, 38.7% intermediate and 3.3% resistant strains with MIC 50=0.06 mg/l and MIC 90=0.12 mg/l. A high proportion (100%) of isolates were fully resistant to Sm, while 22.6% of strains were resistant to Em. In conclusion, this study showed that Cro, Ctx, Cip and Rif were the most efficient drugs. Sulfonamides must be used neither for treatment nor for prophylaxis, as they would be ineffective. Serogroups B and C were predominant in this period in Romania. The need for new antimicrobial agents and vaccines is being felt in today's society.

Quality assessed nonculture techniques for detection and typing of meningococci.

PCR protocols are increasingly used in laboratories worldwide for the diagnosis and confirmation of invasive meningococcal infection. Protocols are now available for the identification of Neisseria meningitidis, for genogrouping, susceptibility to antibiotics and genotyping of the corresponding isolates. The implementation of quality assurance (QA) schemes and standardization of protocols are required. Diagnostic and confirmatory PCRs should perform consistently in clinical and reference microbiology laboratories. General QA schemes address the issues of sample preparation, PCR laboratory environment, equipment and validation of protocols. Moreover, external QA interlaboratory studies are essential. The European Monitoring Group on Meningococci has provided a good forum to conduct such studies through the development and distribution of samples and protocols for nonculture detection and typing of N. meningitidis.

Prospective controlled study of the diagnostic value of skin biopsy in patients with presumed meningococcal disease.

Microbiological tests for diagnosis of acute meningococcal disease are important for the clinical management of patients with this often-fatal illness, but cultures are frequently negative after antibiotics have been administered. Retrospective studies suggest that examination of skin biopsies may aid a rapid diagnosis and that cultures of skin biopsies are often positive even after antimicrobial treatment has commenced. This prospective controlled study aimed to assess the diagnostic value of skin biopsy compared with investigations of blood and cerebrospinal fluid (CSF) in patients with skin lesions and presumed meningococcal disease. A total of 43 patients, 31 with suspected acute meningococcal infection and 12 controls, were included. All skin biopsies were investigated by Gram stain and routine microbiological culture. In 25 patients, meningococcal infection was diagnosed microbiologically. The clinical diagnosis was meningococcal meningitis in 8 patients, meningococcal sepsis in 11 patients, and a combination of both in 6 patients. The sensitivity of cultures of blood, CSF, and skin biopsies was 56%, 50%, and 36%, respectively. When culture and Gram stain were combined, positive results were obtained in 56%, 64%, and 56%, respectively. There was no correlation between the diagnostic yield of skin biopsies and previous antibiotic treatment. In 14 patients, the diagnosis was based exclusively on one positive sample: CSF in 7 (28%) patients, blood in 4 (16%) patients, and skin biopsy in 3 (12%) patients. The sensitivity of skin biopsies was highest in patients with the least extensive skin lesions. Specificity was 100%. Microbiological investigation of skin biopsies increased the diagnostic yield and could be considered a component of the routine diagnostic work-up in patients with suspected meningococcal infection, even after the initiation of antimicrobial treatment.