Age-specific application of neutrophil-to-lymphocyte ratio in meningitis: a nationwide study.

Cerebrospinal fluid (CSF) neutrophil counts and neutrophil-to-lymphocyte ratio (NLR) are useful in distinguishing bacterial and viral meningitis. Given that meningitis is clinically heterogeneous with regard to age, here we investigated the validity of the CSF NLR and neutrophil assay according to age group. Data from the nationwide referral of >4,000 meningitis cases to the Hellenic Meningitis Reference Laboratory between 2006 and 2013 were examined. CSF NLR and neutrophil counts were stratified according to age, and assay performance was determined using previous cut-off values of 2 and 287 cells/µl for CSF NLR and neutrophils respectively. The distribution of bacterial versus viral meningitis was heterogenous across age groups, with a low proportion of bacterial meningitis in patients aged 5-14. CSF neutrophil count and NLR were significantly more discriminatory for bacterial meningitis in patients aged over 14 years than those aged 0-14. The odds ratio (OR), sensitivity, specificity and positive predictive value (PPV) were significantly higher in older patients for both biomarkers. When combined, the false-positive and false-negative detection of bacterial meningitis was 3.9 and 8.5% respectively, and the OR of 262.2 was 2.5-fold greater than expected from a multiplicative effect alone in patients aged >14 years. Care is required when applying diagnostic tests for meningitis in different age groups because of patient heterogeneity. This is the first description of the age distribution of meningitis cases in Greece, and knowledge of the age-related distribution of neutrophils and NLR in meningitis cases could help towards developing age-specific meningitis diagnostic assays.

Neutrophil-to-lymphocyte ratio in the differential diagnosis of acute bacterial meningitis.

The differential diagnosis of acute community-acquired meningitis is of paramount importance in both therapeutic and healthcare-related economic terms. Despite the routinely used markers, novel, easily calculated, and rapidly available biomarkers are needed particularly in resource-poor settings. A promising, exponentially studied inflammatory marker is the neutrophil-to-lymphocyte ratio (NLR), albeit not assessed in meningitis. The aim of this study was to investigate the utility of the NLR in the differential diagnosis of acute meningitis. Data on cerebrospinal fluid (CSF) and blood leukocyte parameters from more than 4,000 patients diagnosed with either bacterial or viral meningitis in Greece during the period 2006-2013 were retrospectively examined. The diagnostic accuracy of the NLR and neutrophil counts in CSF and blood were evaluated by receiver operating characteristic curves. The discrimination ability of both the NLR and neutrophil counts was significantly higher in CSF than in blood. The optimal cutoff values of the NLR and neutrophil counts were 2 in CSF vs 8 in blood, and 287 cells in CSF vs 12,100 cells in blood, respectively. For these values, sensitivity, negative predictive value, and odds ratio were statistically significantly higher in CSF than blood for both markers. Logistic regression analysis showed that the CSF NLR carries independent and additive information to neutrophil counts in the differential diagnosis of acute meningitis. This study is the first one to assess NLR in acute meningitis, providing promising results for its differential diagnosis.

Elevated soluble urokinase receptor values in CSF, age and bacterial meningitis infection are independent and additive risk factors of fatal outcome.

The aim of the present study was to evaluate the potential role of cerebrospinal fluid soluble urokinase receptor (suPAR) level, infection and age as risk factors for fatal outcome in patients suspected of having meningitis and/or bacteraemia on admission to hospital. A total of 545 cerebrospinal fluid samples from patients with clinically suspected meningitis were sent to the Hellenic National Meningitis Reference Laboratory. Ten of 545 (1.83%) patients died. Analysis by receiver operating characteristics (ROC) curve revealed that both suPAR and age were significant for prediction of fatal outcome. Patients with levels of suPAR above the cut-off values and age ≥ 51 years, or patients in which either Neisseria meningitis or Streptococcus pneumoniae were detected were categorized as high risk patients. The combination of the above three predictors (suPAR, age and infectious agent) in a logistic regression model with outcome of infection as the dependent variable yielded an overall odds ratio (OR = 85.7, 95% CI 10.6-690.2) with both sensitivity and specificity being equal to the value of 0.9. In conclusion, suPAR, age and type of infection have an additive effect in predicting mortality among patients suspected of meningitis.

Complicated pneumococcal meningitis in a fully vaccinated child: value of magnetic resonance imaging monitoring.

We present a unique case of life-threatening pneumococcal meningitis complicated by vasculitis in a fully vaccinated 4-year-old female with the heptavalent conjugate vaccine for Streptococcus pneumoniae (PCV7). Serotype 23F was isolated in both blood and cerebral spinal fluid (CSF) samples. Pulses of methylprednisolone were promptly initiated in addition to antibiotics leading to a good recovery. CSF studies and magnetic resonance imaging (MRI) of the brain were used for diagnosis and monitoring the response to corticoid therapy. We report the investigations of a child with failure of PCV7 vaccine to protect against vaccine-serotype invasive disease. Use of corticosteroids and temporal association with changes in brain imaging are described for the first time in literature.

Estimated strain coverage of serogroup B meningococcal vaccines: A retrospective study for disease and carrier strains in Greece (2010-2017).

Invasive meningococcal disease (IMD) is associated with high case fatality rates and long-term sequelae among survivors. Meningococci belonging to six serogroups (A, B, C, W, X, and Y) cause nearly all IMD worldwide, with serogroup B meningococci (MenB) the predominant cause in many European countries, including Greece (~80% of all IMD). In the absence of protein-conjugate polysaccharide MenB vaccines, two protein-based vaccines are available to prevent MenB IMD in Greece: 4CMenB (Bexsero™, GlaxoSmithKline), available since 2014; and MenB-FHbp, (Trumenba™, Pfizer), since 2018. This study investigated the potential coverage of MenB vaccines in Greece using 107 MenB specimens, collected from 2010 to 2017 (66 IMD isolates and 41 clinical samples identified solely by non-culture PCR), alongside 6 MenB isolates from a carriage study conducted during 2017-2018. All isolates were characterized by multilocus sequence typing (MLST), PorA, and FetA antigen typing. Whole Genome Sequencing (WGS) was performed on 66 isolates to define the sequences of vaccine components factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA), and Neisseria adhesin A (NadA). The expression of fHbp was investigated with flow cytometric meningococcal antigen surface expression (MEASURE) assay. The fHbp gene was present in-frame in all isolates tested by WGS and in 41 MenB clinical samples. All three variant families of fHbp peptides were present, with subfamily B peptides (variant 1) occurring in 69.2% and subfamily A in 30.8% of the samples respectively. Sixty three of 66 (95.5%) MenB isolates expressed sufficient fHbp to be susceptible to bactericidal killing by MenB-fHbp induced antibodies, highlighting its potential to protect against most IMD in Greece.

Meningococcal group C disease in Greece during 1993-2006: the impact of an unofficial single-dose vaccination scheme adopted by most paediatricians.

The aim of this study was to evaluate the impact of the meningococcal C conjugate vaccine on the epidemiology of meningococcal C disease in Greece. Data from the National Reference Laboratory for Meningococcal Disease and a questionnaire distributed to Greek paediatricians were assessed. Since the introduction of the vaccine in 2001, 72% of Greek paediatricians have administered it as one single dose to patients aged > or =12 months. This vaccination scheme has probably contributed to a dramatic decrease in the number of meningococcal C infections, which reached zero in 2004.

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece.

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.

Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae.

Rapid, accurate and inexpensive diagnosis of bacterial meningitis is critical for patient management. This study describes the development and evaluation of a multiplex PCR assay for the detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b, which globally account for 90% of cases of bacterial meningitis. The single-tube assay, based on the ctrA, ply and bex targets, respectively, enabled detection of 5-10 pg DNA. When the assay was tested with clinical samples (n = 425), its sensitivity for the three targets was 93.9%, 92.3% and 88%, respectively, while the overall specificity and positive predictive value of the assay was 100%. The negative predictive value was 99.1-99.5%. The methodology permits rapid and accurate detection of the three main pathogens that cause bacterial meningitis.

Serogroups, serotypes and subtypes of Neisseria meningitidis isolated from patients and carriers in Greece.

The increase in the number of cases of meningococcal disease reported to the Ministry of Health in Athens since 1989 prompted the present study to determine if isolates from patients or carriers expressed the same phenotypic characters as those in other parts of Europe. None of the isolates from patients (31) or carriers (547) expressed the antigenic combinations associated with outbreaks in northern Europe, i.e., B:15:P1.16 or B:4:P1.15. The majority of the Greek isolates did not react with any of the six monoclonal serotype reagents tested; however, most reacted with one or more of the 11 monoclonal subtype antibodies. The results suggest that additional serotype reagents are needed for epidemiological studies in southeastern Europe and that vaccines based on serotype antigens developed against outbreak strains in northern Europe would not be effective in Greece.

Reactivity of the new monoclonal antibody '22' with meningococcal strains isolated from patients and carriers in Greece.

Previous studies found that the majority of Neisseria meningitidis isolates from either patients or carriers in Greece do not react with the monoclonal antibodies used at present in the whole-cell ELISA (WCE) for determination of serotype and subtype antigens. A new monoclonal antibody designated '22' produced by the National Meningococcal Reference Laboratory in the Czech Republic was assessed in the whole-cell ELISA with 257 non-typable meningococcal strains from both patients (52) and carriers (205). The carrier strains included 34 non-typable isolates from two immigrant populations: ethnic Greeks who have immigrated from Russia since 1989 (19/75) and Kurdish refugees (15/34). Approximately 10% of the meningococcal strains isolated from patients and 11.7% of the carrier strains reacted with the reagent. Although the majority of meningococcal isolates from resident Greeks were not typable with the antibody, 11/19 (57.9%) of the carrier strains from Russian immigrants and 4/15 (20%) of those from the Kurdish refugees reacted with the new reagent.

Detection of IgG and IgM to meningococcal outer membrane proteins in relation to carriage of Neisseria meningitidis or Neisseria lactamica.

Carriage of non-serogroupable Neisseria meningitidis or Neisseria lactamica induces antibodies protective against meningococcal disease. Antibodies directed against outer membrane proteins are bactericidal and the serotype and subtype outer membrane protein antigens are being examined for their value as vaccine candidates for serogroup B disease. The aim of this study was to examine the effect of carriage of these two Neisseria species among children and young adults on induction of antibodies to outer membrane components from strains causing disease in Greece. Among 53 patients with meningococcal disease, IgG or IgM antibodies were detected by ELISA in 9 of 13 (69%) from whom the bacteria were isolated and 27 of 40 (67%) who were culture-negative. For military recruits (n = 604), the proportion of carriers of meningococci with IgM or IgG to outer membrane proteins was higher than non-carriers, P < 0.05 and P = 0.000000, respectively. Among school children (n = 319), the proportion with IgM or IgG to outer membrane proteins for carriers of meningococci was higher compared with non-carriers, P = 0.000000 and P = 0000043, respectively. Carriage of N. lactamica was not associated with the presence of either IgM or IgG to the outer membrane proteins in the children. The higher proportion of children (50%) with IgM to outer membrane proteins compared with recruits (10%) might reflect more recent exposure and primary immune responses to the bacteria. The lack of association between antibodies to outer membrane proteins and carriage of N. lactamica could reflect observations that the majority of N. lactamica isolates from Greece and other countries do not react with monoclonal typing reagents. Bactericidal antibodies to meningococci associated with high levels of IgG to N. lactamica were found in a previous study; these are thought to be directed to antigens other than outer membrane proteins or capsules and imply antigens such as lipo-oligosaccharide are involved in induction of antibodies cross-reactive with meningococci.

Detection of microbial surface antigens that bind Lewis(a) antigen.

There is evidence that the Lewis(a) blood group antigen is one of the receptors for a number of potentially pathogenic microorganisms. To determine how widely distributed the microbial adhesins are that bind this antigen, anti-idiotypic antibodies produced against monoclonal anti-Lewis(a) were used in coagglutination assays to screen a variety of species. The following were agglutinated: 7/7 strains of Staphylococcus aureus; 10/19 (53%) strains of Neisseria meningitidis; 8/13 (62%) strains of Haemophilus influenzae; 1/3 strains of Helicobacter pylori; 1/2 strains of Neisseria gonorrhoeae; 1/2 strains of Candida albicans. The application of the anti-idiotypic antibodies to studies of host cell receptors, isolation of adhesins and development of new epidemiological typing reagents is discussed.

Carriage of Neisseria meningitidis and Neisseria lactamica among ethnic Greek school children from Russian immigrant families in Athens.

During February and March 1995, a survey of meningococcal carriage in 625 school children was carried out in a suburb of Athens in which there was a large number of ethnic Greeks who had immigrated from Russia beginning in the early 1990s. The objectives of the study were: (1) to determine if factors associated with carriage of meningococci observed in a previous study of Greek school children were similar for the immigrant population; (2) to compare phenotypic characteristics of meningococci from the immigrant population with those isolated from children in Athens. Overall isolation rate for meningococci was 82/625 (13.1%), significantly higher than that found for school children in Athens (5.8%) during the winter of 1990 1991 (5.8%) (chi=25.98, P=0.0000003). By univariate analysis, carriage was not associated with sex, number of individuals per household, blood group, secretor status, socioeconomic level or maternal smoking; however, it was associated with fathers' smoking. The high proportion of men who smoked compared with the low proportion of women smokers might contribute to this finding. The main serogroup of meningococci isolated from this population was A (28%). While serogroup A appears to be more prevalent among Russian and Kurdish immigrants (14%) than among Greek school children or military recruits (4%), there has not been an increase in group A meningococcal disease in Greece. The isolation rate for N. lactamica was high 105/625 (17.3%). A few of these strains bound some of the monoclonal antibodies used for meningococcal serotyping and subtyping, and they are being examined in greater detail.

Recent emergence of serogroup C meningococcal disease in Greece.

The number of cases of meningococcal disease reported to the Meningitis Reference Laboratory in Athens rose dramatically in 1996-1997. The aims were (1) to determine if the increase was due to introduction of new strains, (2) to assess the geographic and age distribution of the cases, (3) to compare antibiotic sensitivity patterns of the current isolates with strains from the early 1990s. In 1993-1994, 15/19 (74%) of the cases for which information on age was available were in children < or = 5 years; in 1995-1997, 80/179 (45%) of cases were in children < or = 5 years and 99 (55%) in the older age range (P < 0.02). From 593 cases in 1993 1997, 214 (36%) isolates were available for characterisation. Serogroup B was predominant in the early 1990s, but by 1997, serogroup C accounted for 46/72 (64%) of isolates and serogroup B for 25/72 (35%). Serogroup B was predominant in children < or = 5 years (44/78, 56%) but only 19/99 (18%) of older children and adults (P=0.0000005). Sulfonamide resistance decreased from 10/22 (45%) in 1993-1994 to 27/192 (14%) in 1995-1997 (P<0.01). Multilocus enzyme electrophoresis of 70 strains obtained during this period identified the epidemic ET-15 clone in 24 (34.3)%. The profiles of the Greek ET-15 isolates were identical to C:2a:P1.2(P1.5) strains responsible for the epidemic in the Czech Republic which began in 1993. This genotype was not found in Greek strains isolated prior to 1993. We conclude that the increase in meningococcal disease is due to introduction of the epidemic serogroup C:2a:P1.2(P1.5) strain responsible for disease in the Czech Republic and Canada.

Cases of Neisseria meningitidis in Greece.

In Greece, Neisseria meningitidis appears as sporadic cases with some seasonal local outbreaks. The notification of meningococcal disease to the local health authorities and the Ministry of Health is mandatory. In 1994, the National Reference Centre for M

Invasive meningococcal disease in children in Greece: comparison of serogroup A disease with disease caused by other serogroups.

Although invasive meningococcal disease caused by serogroup A is not prevalent in developed countries, a considerable number of cases were recently recorded in Greece. In this study, serogroup A meningococcal disease was compared prospectively with meningococcal disease caused by other serogroups, using similar settings of testing and management during a 5-year period between 1999 and 2003. The Neisseria meningitidis serogroup was determined in 262 cases. Serogroup B predominated, accounting for 158 (60%) of the cases. Serogroup A was second most frequent (19%), followed by serogroups W135 (11%), C (8%), and Y (2%). No cases due to serogroup C were recorded during the last year of the study. Patients with serogroup A disease were older and had a milder course compared to patients infected with serogroups B or C. Toxic appearance, purpura, thrombocytopenia, abnormal coagulation tests, and the need for admission to the intensive care unit, fluid resuscitation, inotropic drugs, and mechanical ventilation were less common. Although morbidity and mortality were lower in these patients, the differences were not significant. Serogroup B is predominant in our area, and the introduction of an effective vaccine against it is a priority. Serogroup A has emerged as the second most common serogroup, but the illness associated with it is milder.

Parental smoking and carriage of Neisseria meningitidis among Greek schoolchildren.

In December 1990 and January 1991, primary (320) and secondary (697) pupils in 2 areas of Athens were screened to determine the rate of carriage of Neisseria meningitidis and to determine if the genetic and environmental factors associated with carriage of meningococci in Greece were similar to those observed for northern European populations. In 1 area, socioeconomic indicators were significantly lower than in the other (p < 0.0005), but the isolation rates from pupils in the areas were similar, 5.3% and 6.3%. In contrast to studies in northwest Europe, carriage was not associated with lower socioeconomic conditions, sex, numbers of individuals per household, upper respiratory tract infection, or secretor status. By univariate analysis, carriage was associated with age (15-18 years) (p < 0.05) and mother's or other carer's smoking habits (p < 0.05)--but not father's smoking. Although the proportion of fathers who smoked was greater in the area where socioeconomic indicators were lower (61%) vs. (47%) (p < 0.0005), the proportions of women smokers were similar (33% vs. 38%). By multiple regression analysis, the only significant factors were age (p < 0.01) and carer's smoking (p < 0.05).

Neisseria gonorrhoeae in Athens, Greece. Epidemiologic classification and antimicrobial susceptibility patterns of strains isolated between 1986 and 1989.

The incidence of gonorrhoea cases diagnosed in a major venereal hospital of Athens, Greece, was markedly reduced between 1986 and 1989. All 182 gonococcal isolates found during this period were epidemiologically classified into Sero-/Auxo-/Plasmid content (S/A/Pc) types. Susceptibility to six antimicrobials was also determined. Penicillinase-producing Neisseria gonorrhoeae (PPNG) strains were isolated in an increased frequency of 7.7% and a striking shift in their distribution into S/A/Pc types was noted from previous years. The isolates exhibited wide heterogeneity of type: none of the 14 PPNG strains was identical, while the 168 non-PPNG strains were distributed into 64 S/A/Pc types. Fifty-seven percent (57%) of the PPNG and 86% of the non-PPNG isolates were of the WII/III serogroup. Chromosomally mediated resistance to penicillin, ampicillin, or both was exhibited by 14% of the non-PPNG isolates. Greater frequency of reduced susceptibility to cefotaxime, tetracycline, and chloramphenicol was also found among PPNG and non-PPNG strains, mostly of the WII/III serogroup. Twelve percent (12%) of the non-PPNG and 57% of the PPNG strains had multiple patterns of resistance, involving 2 to 4 antimicrobials of different class lines.

Antibiotic sensitivities of Neisseria meningitidis isolates from patients and carriers in Greece.

Usage of antibiotics in southern Europe is less well regulated than in northern countries. The proportion (48%) of meningococci in Spain insensitive to pencillin (MIC greater than or equal to 0.1 mg/l) prompted this investigation of antibiotic sensitivities of isolates from Greek patients with meningitis (31) and carriers (47 school-children and 472 recruits). The agar dilution method was used to determine MIC to penicillin G (PN), sulphamethoxazole (SU), rifampicin (RF), cefaclor (CF) and ciprofloxacin (CP). The proportion of isolates insensitive to PN was 48% for isolates from patients, 19% from school-children and 36.6% from recruits. Resistance to SU (MIC greater than or equal to 16 mg/l) was found in 16% of those from patients, 10.6% from children and 40% from recruits. None of the isolates from patients was resistant to RF (greater than or equal to 1 mg/l) but 6% of those from carriers were. Resistance to CF (greater than or equal to 4 mg/l) was found in 9.2% of patient isolates, 6.4% from children and 23.7% from recruits. All isolates except one were sensitive to CP (MIC range less than 0.0015-0.125 mg/l). Resistances to PN, SU and RF were analysed by serogroup, serotype and subtype of the bacteria. The proportion of resistant isolates showed some variation between different areas of Greece, but it was not statistically significant.

Antibiotic resistance, plasmid content and auxotypes of Neisseria gonorrhoeae in Greece.

The antibiotic resistance pattern, the plasmid content and the auxotypes of 27 PPNG and 200 non-PPNG isolates obtained in 1984-1985 were examined. The well-known plasmid patterns, the most frequent was the combination of 4.5 and well-known plasmid patterns, the most frequent was the combination of 4.5 and 2.6 Mdal plasmids. The strains with the Asian-type (4.5 Mdal) plasmid belonged to the auxotypes Pro and Zero, whereas those with the African type (3.2 Mdal) plasmid were of auxotypes Zero, Arg and Pro, Arg. All PPNG strains exhibited reduced sensitivity at least to one of the other antibiotics tested (cefotaxime, spectinomycin, thiamphenicol, tetracycline). Among non-PPNG strains, 19% harboured the conjugative and the cryptic plasmid, 67.5% only the cryptic, 1% only the conjugative and 12% were plasmid-free. The most frequent auxotypes of non-PPNG strains were Zero (65.5%) and Pro (21%), while the uncommon auxotype Pro, Arg degree, Ura was found only in plasmid-free strains with reduced sensitivity to penicillin. Susceptibility testing revealed a large number of non-PPNG strains with decreased sensitivity to antibiotics, the Pro and Zero isolates being less sensitive than those of other auxotypes.