A multicenter study on the epidemiology of complicated parapneumonic effusion in the era of currently available pneumococcal conjugate vaccines.

BACKGROUND: Parapneumonic effusion (PPE) is a common complication of pneumonia. Streptococcus pneumoniae is the most common cause of bacterial pneumonia. A reduction in pneumonia hospitalizations has been observed since the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7). Despite this apparent benefit, an increase in the incidence of PPE was recorded in some countries following PCV7 implementation. As the 13-valent pneumococcal conjugate vaccine (PCV13) was expected to provide a wider protection against PPE, the aim of the present study was to evaluate the impact of PCV13 introduction on the epidemiology of complicated parapneumonic effusion (c-PPE) among children in the Athens greater area. METHODS: All cases of community-acquired pneumonia (CAP) with PPE requiring chest tube insertion (complicated PPE, c-PPE) hospitalized in the 3 public Children's hospitals in Athens between 01/01/2004 and 31/12/2019 were included in the study. RESULTS: A total of 426 cases of c-PPE associated with pneumonia were recorded of which 198 were admitted during 2004-2010 (period A, prePCV13/PCV -7 introduction period) and 228 during 2011-2018 (period B, post - PCV13 period). A definite bacterial etiology was established in 44.4 % of all cases and of those 25.4 % were caused by S. pneumoniae. An increasing trend in c-PPE incidence was observed during period A; although, a significant decrease on c-PPE annual rates was observed during the period B (p = 0.011), a remarkable increase in serotype 3 cases was recorded. CONCLUSION: A decreasing time trend in c-PPE cases among children was shown after the introduction of PCV13 in our area. However, serotype 3 is nowadays a common cause of PPE. Hence, continuous surveillance is imperative in order to follow c-PPE epidemiology over time.

Meningococcal Antibiotic Resistance: Molecular Characterization of Isolates from Patients with Invasive Meningococcal Disease (IMD) in Greece.

For effective case management and chemoprophylaxis of Invasive Meningococcal Disease (IMD), prompt antibiotic treatment is required. N. meningitidis is usually susceptible to antibiotics, but reduced susceptibility to penicillin, ciprofloxacin, and rifampicin is increasing worldwide, jeopardizing patients' outcome. We assessed, phenotypically and genotypically, the antimicrobial resistance patterns of 192 strains isolated from IMD cases from all over Greece during 2010-2021. Antimicrobial susceptibility to penicillin, rifampicin, and ciprofloxacin was determined using the E-test. All isolates were genotyped by Multilocus Sequence Typing (MLST). penA, rpoB, and gyrA genes were amplified by PCR and sequenced. Of the 192 isolates, 37% (72/192) were penicillin-susceptible/had increased exposure, and 11% (21/192) were penicillin-resistant. Among those, 40 penA alleles were identified; penA1, penA27, and penA3 were highly associated with susceptibility to penicillin; penA14, penA25, and penA22 related to reduced susceptibility to penicillin, while penA9, penA910, and penA295 had resistance to penicillin. Two ciprofloxacin-resistant isolates harbored the gyrA346 allele, while one rifampicin-resistant isolate harbored the rpoB5 allele. Resistance to ciprofloxacin and rifampicin remains rare. As Greece is one of the countries with high antimicrobial resistance, continued monitoring of antibiotic resistance is important to ensure timely detection of emerging resistance for treatment and prevention guidelines.

Impact of a Single-Tube PCR Assay for the Detection of Haemophilus influenzae Serotypes a, c, d, e and f on the Epidemiological Surveillance in Greece.

Background: The decrease in the rate of meningitis due to Haemophilus influenzae type b after vaccine introduction and a possible change in epidemiology of H. influenzae disease highlights the need for continuous serotype surveillance. Methods: A single-tube multiplex PCR assay for serotyping of H. influenzae was developed and deployed. Results: During 2003−2020, 108 meningitis cases due to H. influenzae were notified; 86 (80%) were confirmed and serotyped by molecular methods. The overall specificity and sensitivity of the assay were estimated (100% PPV and NPV respectively). The overall mean annual reported incidence for H. influenzae was 0.02, while for Hib and non-b meningitis equaled 0.02 and 0.03 per 100 000, respectively. Analysis by age group revealed that H. influenzae peaks in toddlers and children 0−4 years and in adults >45 years old. Among the serotyped cases, 39.8% were identified as Hib, 46.3% as NTHi, and 0.9% and 2.8% as serotypes a (Hia) and f (Hif)) respectively. Conclusions: Low incidence due to Hib was observed while non-typeable H. influenzae (NTHi) and serotypes Hia and Hif seem to emerge. The application of the current assay discloses the ongoing change of invasive H. influenzae disease trends during the Hib post-vaccine era.

Pneumococcal meningitis in Greece: A retrospective serotype surveillance study in the post-PCV13 era (2010-2020).

BACKGROUND: As Greece is a country which has introduced the 13-valent pneumococcal conjugate vaccine (PCV13) both in the infant and in the adult immunization programs, the aim of the study was to investigate age-specific and serotype-specific trends of pneumococcal meningitis over an 11-year period (2010-2020). MATERIALS AND METHODS: Data are reported from pneumococcal meningitis cases [notified to the National Public Health Organization (NPHO)], with clinical samples and bacterial isolates sent for pneumococcal identification and serotyping at the National Meningitis Reference Laboratory (NMRL). Pneumococcal identification was performed directly on clinical samples or bacterial isolates by multiplex PCR (mPCR) assay, while serotyping was carried out by application of the Capsular Sequence Typing (CST) method with the combination of single tube PCR assays. RESULTS: A total of 427 pneumococcal meningitis cases were notified to the NPHO between 2010 and 2020. Among those, 405 (94.8%) were microbiologically confirmed, while samples from 273 patients were sent to the NMRL for identification and/or further typing. The annual notification rate peaked at 0.47/100,000 in 2016 and since then has been decreasing. The incidence was highest in infants and in older adults. Pneumococcal serotypes were identified in 260/273 (95.2%) cases, where clinical samples were sent to the NMRL. The most prevalent serotypes (≥5%) were 3, 19A, 23B, 15B/C, 11A/D, 23A, 22F. During the study period there has been a decrease of PCV13 serotypes combined with an increase of non-PCV13 serotypes (p = 0.0045). CONCLUSIONS: This is the first study to report serotypes for pneumococcal meningitis across all ages in the post-PCV13 era in Greece. There is a need to enhance surveillance, by close monitoring of the emerging serotypes and the impact of vaccination programs. Higher-valency PCVs may help to improve the coverage of pneumococcal disease.

Pneumococcal Serotype Identification by Capsular Sequence Typing (CST): A Modified Novel Approach for Serotyping Directly in Clinical Samples.

As almost 60-70% of Invasive Pneumococcal Disease (IPD) is identified by nonculture methods in Greece, serotyping is of high importance for the better monitoring of pneumococcal serotypes due to the availability of conjugate vaccines. The aim of the study was the modification and direct application of the Capsular Sequence Typing (CST) assay in clinical samples in order to serotype Streptococcus pneumoniae culture-negative, Polymerase Chain Reaction (PCR_-positive samples, followed by CST group specific single-tube PCR assays. A two-step PCR modified assay was applied on a total of 306 samples (such as CSF, blood, pleural and middle ear fluids, isolates) obtained from 283 patients with IPD. The overall performance permits a rapid, accurate and cost-effective method for nonculture pneumococcal serotyping. As the management of IPD is closely related to the continuous monitoring of pneumococcal serotypes, the proposed approach proved to be a valuable tool for the typing and epidemiological monitoring of S. pneumoniae, for the evaluation of the overall impact of vaccination programs in the era of pneumococcal conjugate vaccines, in order to initiate the appropriate vaccination strategy.

Symptomatic Female Genital Tract Infections Due to Neisseria meningitidis in Athens, Greece.

Neisseria meningitidis is considered as an obligate human pathogen and can cause life-threatening diseases like meningitis and/or septicaemia. Occasionally, it can be recovered from infections outside the bloodstream or central nervous system, like respiratory, ocular, joint, urogenital or other unusual sites. Herein, we present two rare cases of female genital infections due to N. meningitidis within a two-year period (2019-2020), identified as serogroup B (MenB) and Y (MenY), respectively. Genotypic analysis for PorA, FetA and MLST revealed the following characteristics: MenB: 7-12, 14, F5-36, 1572cc and MenY: 5-1,10-1, F4-5, 23cc, respectively. Such unusual presentations should alert the clinicians and microbiologists not to exclude N. meningitidis from routine diagnosis and the need of early detection. This is the first report in Greece, and, to our knowledge, in Europe since 2005 describing meningococcal female genital infections.

Artificial Intelligence in Differential Diagnostics of Meningitis: A Nationwide Study.

Differential diagnosis between bacterial and viral meningitis is crucial. In our study, to differentiate bacterial vs. viral meningitis, three machine learning (ML) algorithms (multiple logistic regression (MLR), random forest (RF), and naïve-Bayes (NB)) were applied for the two age groups (0-14 and >14 years) of patients with meningitis by both conventional (culture) and molecular (PCR) methods. Cerebrospinal fluid (CSF) neutrophils, CSF lymphocytes, neutrophil-to-lymphocyte ratio (NLR), blood albumin, blood C-reactive protein (CRP), glucose, blood soluble urokinase-type plasminogen activator receptor (suPAR), and CSF lymphocytes-to-blood CRP ratio (LCR) were used as predictors for the ML algorithms. The performance of the ML algorithms was evaluated through a cross-validation procedure, and optimal predictions of the type of meningitis were above 95% for viral and 78% for bacterial meningitis. Overall, MLR and RF yielded the best performance when using CSF neutrophils, CSF lymphocytes, NLR, albumin, glucose, gender, and CRP. Also, our results reconfirm the high diagnostic accuracy of NLR in the differential diagnosis between bacterial and viral meningitis.

First report of meningococcal ciprofloxacin resistance in Greece due to invasive isolates of the sequence type ST-3129.

A local outbreak caused by Neisseria meningitidis occurred in the migration camp in the Greek island of Lesbos during January-February 2020 (4 of 5 cases). In total, 5 samples positive for N. meningitidis were further investigated for sero-/genogroup, PorA, and WGS analysis. MenB was found among 3 cases, while in two cases, MenY was identified. WGS analysis and antibiotic susceptibility testing on the 2 culture positive MenB samples showed the new ST-3129, ciprofloxacin-resistant clone was circulating among the immigrants in the aforementioned camp. This is the first report of ciprofloxacin resistance in Greece.

Epidemiology of invasive meningococcal disease in Greece, 2006-2016.

The present study describes the epidemiology of invasive meningococcal disease (IMD) in Greece for the period 2006-2016. Combined data from notified and laboratory-confirmed IMD cases were obtained from the two involved National Centres (Epidemiology and Reference Laboratory). Laboratory identification and typing was carried out by both conventional (culture) and molecular methods (PCR, MLST, PorA, and FetA typing). A total of 796 IMD cases were notified; of those, 720 (91%) were laboratory confirmed. Overall, a decline on the annual incidence of confirmed cases was observed, ranging from 0.91 (2006) to 0.47 (2016) /100,000. A similar trend was observed in most age groups especially in children 0-4 years (7.7 to 2.9/100,000), with the exception of an increase in the incidence rate in adults > 20 years (0.21 to 0.32/100,000). The overall case fatality rate was 6.5% (52/796), annual range 2-13%. Among 658 strains which were typed by sero/genogroup, 80% were identified as MenB (annual range 65-92%); however, a decline was observed in MenB incidence from 5.3 (2006) to 2.7 (2016), among infants and toddlers, while MenW (1%), MenY (2%), and MenA (1%) remained low. During the 11 years, the annual incidence of IMD declined by 50%, especially in the 0-4-year age group, due mainly to MenB. Continuous surveillance of IMD is important for the development of future vaccination and public health policies.

Characterization of meningococcal carriage isolates from Greece by whole genome sequencing: Implications for 4CMenB vaccine implementation.

Herd protection, resulting from the interruption of transmission and asymptomatic carriage, is an important element of the effectiveness of vaccines against the meningococcus. Whilst this has been well established for conjugate polysaccharide vaccines directed against the meningococcal capsule, two uncertainties surround the potential herd protection provided by the novel protein-based vaccines that are used in place of serogroup B (MenB) polysaccharide vaccines (i) the strain coverage of such vaccines against carried meningococci, which are highly diverse; and (ii) the generation of a protective immune response in the mucosa. These considerations are essential for realistic estimates of cost-effectiveness of new MenB vaccines. Here the first of these questions is addressed by the whole genome sequence (WGS) analysis of meningococci isolated from healthy military recruits and university students in Greece. The study included a total of 71 MenB isolates obtained from 1420 oropharyngeal single swab samples collected from military recruits and university students on voluntary basis, aged 18-26 years. In addition to WGS analysis to identify genetic lineage and vaccine antigen genes, including the Bexsero Antigen Sequence Type (BAST), the isolates were examined with the serological Meningococcal antigen Typing System (MATS) assay. Comparison of these data demonstrated that the carried meningococcal population was highly diverse with 38% of the carriage isolates showed expression of antigens matching those included in the 4CMenB vaccine. Our data may suggest a limited potential herd immunity to be expected and be driven by an impact on a subset of carriage isolates.

Meningococcal Carriage in Military Recruits and University Students during the Pre MenB Vaccination Era in Greece (2014-2015).

PURPOSE: The aim of the study was to estimate the meningococcal carriage rate and to identify the genotypic characteristics of the strains isolated from healthy military recruits and university students in order to provide data that might increase our understanding on the epidemiology of meningococcus and obtain information which helps to evaluate the potential effects on control programs such as vaccination. METHODS: A total of 1420 oropharyngeal single swab samples were collected from military recruits and university students on voluntary basis, aged 18-26 years. New York City Medium was used for culture and the suspected N. meningitidis colonies were identified by Gram stain, oxidase and rapid carbohydrate utilization tests. Further characterisation was carried out by molecular methods (multiplex PCR, MLST, WGS). RESULTS: The overall carriage rate was of 12.7%; 15% and 10.4% for recruits and university students respectively. MenB (39.4%) was the most prevalent followed by MenY (12.8%) and MenW (4.4%). Among the initial 76 Non Groupable (NG) isolates, Whole Genome Sequence Analysis (WGS) revealed that 8.3% belonged to MenE, 3.3% to MenX and 1.1% to MenZ, while, 53 strains (29.4%) were finally identified as capsule null. Genetic diversity was found among the MenB isolates, with 41/44 cc and 35 cc predominating. CONCLUSION: Meningococcal carriage rate in both groups was lower compared to our previous studies (25% and 18% respectively) with predominance of MenB isolates. These findings, help to further our understanding on the epidemiology of meningococcal disease in Greece. Although the prevalence of carriage seems to have declined compared to our earlier studies, the predominant MenB clonal complexes (including 41/44cc and 35cc) are associated with invasive meningococcal disease.

Diversity of Greek meningococcal serogroup B isolates and estimated coverage of the 4CMenB meningococcal vaccine.

BACKGROUND: Serogroup B meningococcal (MenB) isolates currently account for approximately 90% of invasive meningococcal disease (IMD) in Greece with ST-162 clonal complex predominating. The potential of a multicomponent meningococcal B vaccine (4CMenB) recently licensed in Europe was investigated in order to find whether the aforementioned vaccine will cover the MenB strains circulating in Greece. A panel of 148 serogroup B invasive meningococcal strains was characterized by multilocus sequence typing (MLST) and PorA subtyping. Vaccine components were typed by sequencing for factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA) and Neisseria adhesin A (NadA). Their expression was explored by Meningococcal Antigen Typing System (MATS). RESULTS: Global strain coverage predicted by MATS was 89.2% (95% CI 63.5%-98.6%) with 44.6%, 38.5% and 6.1% of strains covered by one, two and three vaccine antigens respectively. NHBA was the antigen responsible for the highest coverage (78.4%), followed by fHbp (52.7%), PorA (8.1%) and NadA (0.7%). The coverage of the major genotypes did not differ significantly. The most prevalent MLST genotype was the ST-162 clonal complex , accounting for 44.6% of the strains in the panel and with a predicted coverage of 86.4%, mainly due to NHBA and fHbp. CONCLUSIONS: 4CMenB has the potential to protect against a significant proportion of Greek invasive MenB strains.

Parapneumonic pleural effusions caused by Streptococcus pneumoniae serotype 3 in children immunized with 13-valent conjugated pneumococcal vaccine.

During 2012, Streptococcus pneumoniae serotype 3 was identified by polymerase chain reaction in 15 out of 20 (75%) pleural fluid specimens from children with pneumococcal pneumonia complicated by parapneumonic pleural effusion in Greece. One-third of these children had been immunized with the 13-valent conjugated pneumococcal vaccine after the age of 12 months, according to the national immunization schedule.

Microbiology of acute mastoiditis and complicated or refractory acute otitis media among hospitalized children in the postvaccination era.

In the post-heptavalent pneumococcal conjugate vaccine era, Streptococcus pneumoniae remains the leading cause of acute mastoiditis and other complicated or refractory acute otitis media among hospitalized children in our settings. Serotype 19A is predominant, invasive and multidrug resistant causing more than half of all mastoiditis cases, two-thirds of cases with subperiosteal abscess and all those requiring mastoidectomy. Continuous surveillance is required.

Comparison of cytokine gene polymorphisms among Greek patients with invasive meningococcal disease or viral meningitis.

High levels of pro-inflammatory cytokines are implicated in the severity of invasive meningococcal disease (IMD) and viral meningitis (VM). This study compared single-nucleotide polymorphisms (SNPs) in pro- and anti-inflammatory cytokine genes among patients with VM or IMD. Patient DNA samples were prepared by the National Meningitis Reference Laboratory in Athens: n=98 for IMD and n=53 for VM. The results for both patient groups were compared with data published for healthy Greek control data. Real-time PCR was used to assess the interleukin (IL) gene SNPs IL6 G-174C, IL1B C-511T, IL1RN T+2018C, IL10 G-1082A and IL8 A-251T and the tumour necrosis factor α (TNF-α) SNP TNFA G-308A. Differences were compared by Fisher's exact test. The genotype for high IL-6 responses was predominant among IMD (51%, P=0.0008) and VM (74.5%, P<0.0001) patients compared with the controls (31%). The genotype associated with high TNF-α responses was 5% among controls and lower for IMD (1.1%, P=0.0014) and VM (0%, P=0.052). There was no difference for IL-8 SNPs between controls and IMD (P=0.162), but the difference was significant for VM (P=0.0025). IL-6 (P=0.024) and IL-8 (P=0.00004) SNPs differed between IMD and VM. Reports on associations between IL-8 SNPs and cytokine responses differ. Because of its role in neutrophil attraction, differences in frequencies of the IL-8 SNP for IMD and VM require further investigation.

Diagnosis of Upper and Lower Respiratory Tract Bacterial Infections with the Use of Multiplex PCR Assays.

The investigation of respiratory infections by molecular techniques provides important information about the epidemiology of respiratory disease, especially during the post-vaccination era. The objective of the present study was the detection of bacterial pathogens directly in clinical samples from patients with upper and lower respiratory tract infections using multiplex polymerase chain reaction (PCR) assays developed in our laboratory. Clinical samples taken over a three-year period (2007-2009) and obtained from 349 patients (adults (n = 66); children (n = 283)) with signs and symptoms of certain upper or lower respiratory tract infections, consisted of: bronchoalveolar lavages (BAL, n = 83), pleural fluids (n = 29), and middle-ear aspirates (n = 237). Overall, 212 samples (61%) were confirmed by culture and/or PCR. Among the positive samples, Streptococcus pneumoniae (mainly serotype 3) was predominant (104/212; 49.0%), followed by non-typable Haemophilus influenzae (NTHi) 59/212; 27.8%) and Streptococcus pyogenes (47/212; 22%). Haemophilus influenzae type b was detected in only three samples. The underlying microbiology of respiratory infections is gradually changing in response to various selective pressures, such as vaccine use and antibiotic consumption. The application of multiplex PCR (mPCR) assays is particularly useful since it successfully identified the microorganisms implicated in acute otitis media or lower respiratory tract infections in nearly 75% of patients with a positive result compared to conventional cultures. Non-culture identification of the implicated pneumococcal serotypes is also an important issue for monitoring pneumococcal infections in the era of conjugate pneumococcal vaccines.

Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples.

This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.