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.

Emergence of OXA-162 Carbapenemase- and DHA-1 AmpC Cephalosporinase-Producing Sequence Type 11 Klebsiella pneumoniae Causing Community-Onset Infection in Greece.

OXA-48-like carbapenemases have only recently emerged in Europe. OXA-162 is a rare OXA-48 variant usually coexpressed with extended-spectrum ß-lactamases. Here, we report the identification of the first OXA-162 carbapenemase-producing Klebsiella pneumoniae isolates, which coexpressed an AmpC cephalosporinase (DHA-1), retrieved from a patient in Greece. They belonged to a single sequence type (ST11) and caused the first documented community-onset urinary tract infections attributable to an OXA-48-like-producing Enterobacteriaceae strain.

Neuroinvasive phlebovirus infection in Greece: a case report.

OBJECTIVE: Sandfly fever phleboviruses are endemic in Mediterranean countries. We report a febrile phlebovirus case in a Greek patient who presented signs of neuroinvasive infection. METHODS: In summer 2010, a 20-year-old male was admitted to hospital with fever and lethargy; he was a resident of central Macedonia, northern Greece, where a large outbreak of West Nile virus (WNV) infections occurred at that time. Since there was no laboratory evidence of WNV infection, the patient's serum and cerebrospinal fluid were tested for a probable phlebovirus infection. RESULTS: High titers of IgM and IgG antibodies against Toscana virus were detected in serum and cerebrospinal fluid, while the titers against sandfly fever Naples virus were lower; no reactivity was detected against sandfly Sicilian and Cyprus viruses. Since neutralization assays were not performed and PCR resulted in being negative, it was concluded that the causative agent was a phlebovirus of the sandfly fever Naples serocomplex. CONCLUSION: The present case confirms results from previous seroprevalence studies showing that phleboviruses of the sandfly fever Naples serocomplex are present in Greece and provides evidence that they cause febrile neuroinvasive disease in humans, prompting for inclusion of phleboviral infections in the differential diagnosis of acute febrile cases during the time when sandflies are active.

Outbreak caused by an ertapenem-resistant, CTX-M-15-producing Klebsiella pneumoniae sequence type 101 clone carrying an OmpK36 porin variant.

Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum ß-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining ß-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.

Imported Klebsiella pneumoniae carbapenemase-producing K. pneumoniae clones in a Greek hospital: impact of infection control measures for restraining their dissemination.

The recent emergence of carbapenemase-producing Enterobacteriaceae strains represents a major threat for hospitalized patients. We document the dissemination and control of carbapenemase-producing Klebsiella pneumoniae clones in a Greek hospital. During a 3-year study period (January 2009 to December 2011), carbapenemase-producing K. pneumoniae strains were isolated from clinical samples from 73 individual patients. Phenotyping and molecular testing confirmed that 52 patients were infected with K. pneumoniae carbapenemase 2 (KPC-2) producers, 12 were infected with VIM-1 producers, and the remaining 9 were infected with isolates producing both KPC-2 and VIM-1 enzymes. Twenty-eight of these clinical cases were characterized as imported health care associated, and 23 of these were attributed to KPC producers and 5 were attributed to KPC and VIM producers. The remaining 45 cases were deemed hospital acquired. In the second year of the study, intensified infection control intervention was implemented, followed by active surveillance and carrier isolation in the third year. The incidence of carbapenemase-producing K. pneumoniae patient cases decreased from 0.52/1,000 patient days in 2009 to 0.32/1,000 patient days in 2010 (P = 0.075). Following these additional infection control measures, the incidence fell to 0.21/1,000 patient days in 2011 and differed significantly from that in 2009 (P = 0.0028). Despite the fact that the imported cases of carbapenemase-producing K. pneumoniae were equally distributed over this 3-year period, the incidence of hospital-acquired cases decreased from 0.36/1,000 patient days in 2009 to 0.19/1,000 patient days in 2010 (P = 0.058) and to 0.1/1,000 patient days in 2011 (P = 0.0012). Our findings suggest that rigorous infection control measures and active surveillance can effectively reduce the incidence of secondary transmission due to KPC-producing pathogens.

Dissemination of clinical isolates of Klebsiella oxytoca harboring CMY-31, VIM-1, and a New OXY-2-type variant in the community.

The aim of the present study was to investigate the epidemiological link of multidrug-resistant Klebsiella oxytoca isolates causing community-onset infections among patients attending our outpatient department and to investigate the underlying resistance mechanisms. The isolates were tested by agar dilution MICs, phenotypic carbapenemase testing, enterobacterial repetitive intergenic consensus-PCR, and pulsed-field gel electrophoresis (PFGE). PCR assays and nucleotide sequencing were employed for the identification of bla gene types and the mapping of the integron-containing metallo-ß-lactamase (MBL) gene. During the study period (January 2005 to April 2007), nine broad-spectrum cephalosporin-resistant K. oxytoca clinical isolates were prospectively collected from separate outpatients with urinary tract infections. In all cases, the patients had been hospitalized or exposed to health care facilities during the preceding year. Molecular typing revealed that all isolates belonged to the same K. oxytoca clonal type, which contained five PFGE subtypes. A novel chromosomal OXY-2 ß-lactamase type variant (OXY-2-9) was detected in all isolates, but no mutations in the promoter region justifying bla(OXY) gene overproduction were detected. In addition, all isolates harbored the plasmidic CMY-31 (LAT-4) AmpC cephalosporinase, while three of them harbored VIM-1 MBL in a class 1 integron structure. This is the first study to present the dissemination in the community of multidrug-resistant K. oxytoca isolates causing extrahospital infections.

Recurrent healthcare-associated community-onset infections due to Klebsiella pneumoniae producing VIM-1 metallo-beta-lactamase.

OBJECTIVES: To investigate the extrahospital dissemination of carbapenem-resistant Klebsiella pneumoniae isolates and the mechanisms of acquired resistance. METHODS: Patients who were referred to the outpatient department of Serres General Hospital with community-onset infections due to carbapenem-resistant K. pneumoniae isolates during August 2007-October 2008 were included in the study. The selected isolates were tested by determination of agar dilution MICs, phenotypic carbapenemase testing and PFGE. PCR and sequencing analyses were employed for identification of bla genes and mapping of the integron carrying the metallo-ß-lactamase (MBL) gene. The location of the MBL allele was investigated by mating experiments, plasmid analysis and PCR assays. RESULTS: Twenty-four carbapenem-resistant K. pneumoniae isolates causing urinary tract infections were recovered from 12 outpatients. Six of the patients presented with recurrent infections within a period of 1-6 months after the initial extrahospital isolation. All patients reported prior hospitalization within the preceding 4 months, whilst two were infected by carbapenem-resistant K. pneumoniae isolates during their previous hospitalization. Imipenem, meropenem and ertapenem MICs ranged from 8 to 64 mg/L, 4 to 32 mg/L and 8 to 128 mg/L, respectively. All studied isolates as well as those obtained from prior hospitalization belonged to a single PFGE clone. They harboured a plasmid-mediated bla(VIM-1) gene in an integron structure that has been previously described among K. pneumoniae isolates causing hospital-acquired infections in Greece. CONCLUSIONS: This is the first study to document the dissemination of an MBL-producing K. pneumoniae strain in the community. The successful strain caused recurrent community-onset infections and was most likely acquired during patients' previous hospitalization.

Large dissemination of VIM-2-metallo-{beta}-lactamase-producing pseudomonas aeruginosa strains causing health care-associated community-onset infections.

During a 3-year period (May 2005 to April 2008), a series of 45 outpatients presented with community-onset urinary tract infections due to carbapenem-resistant Pseudomonas aeruginosa isolates. Forty of them had a history of previous hospitalization or exposure to healthcare facilities, while the remaining five had not been previously admitted to our healthcare facilities or elsewhere within the preceding 12 months. In 18 outpatients, the carbapenem-resistant organisms caused recurrent community-onset urinary tract infections, while in three outpatients the organisms were also implicated in bacteremic episodes. All 45 single-patient P. aeruginosa isolates harbored the bla(VIM-2) metallo-beta-lactamase (MBL) gene in a common class 1 integron structure. They belonged to one predominant pulsed-field gel electrophoresis type and three sporadically detected types; two of the sporadic clonal types were identified among outpatients without previous exposure to healthcare facilities, while the predominant clonal type was also identified to cause infections in hospitalized patients. This is the first study documenting that MBL-producing P. aeruginosa isolates cause community-onset infections that are related or not with exposure to healthcare facilities. Community-onset infections in our patients most likely resulted from the nosocomial acquisition of MBL producers, followed by a prolonged digestive carriage. The high rate of recurrent infections in the community underlies the difficulty of constraining infections caused by such microorganisms in the extrahospital setting.

First occurrence of KPC-2-possessing Klebsiella pneumoniae in a Greek hospital and recommendation for detection with boronic acid disc tests.

OBJECTIVES: To investigate the first KPC carbapenemase-producing Klebsiella pneumoniae isolate from a Greek hospital, including phenotypic methods to aid recognition of this resistance type. METHODS: A carbapenem-resistant clinical isolate of K. pneumoniae was recovered from a hospitalized Greek patient. Detailed susceptibility testing was carried out by the agar dilution method. The isolate was screened by phenotypic and genotypic assays for the presence of various beta-lactamases. Boronic acid disc tests were performed to show the ability of these tests to detect production of the KPC enzymes. The potential for conjugal transfer of carbapenem resistance was examined by biparental matings, plasmid analysis and PCR studies. RESULTS: The isolate possessed on the same self-transferable plasmid the KPC-2 carbapenemase and the SHV-12 extended-spectrum beta-lactamase. Although the isolate did not produce an AmpC-type enzyme, the production of KPC-2 was associated with positive boronic acid disc tests using cephamycins and cefotaxime as well as cefepime and carbapenems as substrates. DISCUSSION: KPC-2-possessing K. pneumoniae clinical isolates seem to have been introduced in our region. Boronic acid disc tests using boronic acid in combination with carbapenems or cefepime may help the phenotypic detection of KPC enzymes and their distinction from plasmid-mediated AmpC enzymes.

Escherichia hermannii as the sole isolate from a patient with purulent conjunctivitis.

Escherichia hermannii was isolated in pure culture from a patient with acute purulent conjunctivitis after a minor ocular injury. This is the first report of E. hermannii isolated as the sole pathogen from an infected site without prior antibiotic exposure, confirming the pathogenic potential of the microorganism.

Transmission in the community of clonal Proteus mirabilis carrying VIM-1 metallo-beta-lactamase.

OBJECTIVES: Several infections among patients attending our outpatient clinic were caused by imipenem-resistant Proteus mirabilis that were phenotypically metallo-beta-lactamase (MBL)-positive. The aim of the study was to investigate this extrahospital dissemination and the mechanisms of resistance to carbapenems. METHODS: During a 1 year period (December 2005-December 2006), the characteristics of the outpatients with infections caused by isolates of P. mirabilis with reduced susceptibility to imipenem (MIC > 4 mg/L) were prospectively collected. The isolates were tested by agar dilution MICs, phenotypic MBL testing and enterobacterial repetitive intergenic consensus PCR. PCR assays and nucleotide sequencing were used for the identification of bla gene types and mapping of the integron carrying the MBL gene. The location of the MBL allele was investigated by mating experiments, plasmid analysis and hybridization of the Southern-blotted plasmid extract with a bla(VIM-1) probe. RESULTS: During the study, 12 MBL-positive P. mirabilis isolates were recovered from urinary tract infections of community patients. In all cases, the patients had a previous hospitalization in a Greek regional hospital and had received fluoroquinolones and/or aminoglycosides and beta-lactams. MICs of imipenem ranged from 32 to >128 mg/L, whereas those of meropenem ranged from 1 to 8 mg/L and those of ertapenem ranged from 0.5 to 4 mg/L. The isolates originated from the same clonal strain and harboured a bla(VIM-1) gene in a common integron structure. Conjugation experiments, plasmid analysis and hybridization assays indicated the chromosomal location of the bla(VIM-1) gene. CONCLUSIONS: This is the first study that documents transmission in the extrahospital setting of acquired MBL-producing Gram-negatives causing community-onset infections.

Emergence of carbapenem-resistant Enterobacter cloacae carrying VIM-4 metallo-beta-lactamase and SHV-2a extended-spectrum beta-lactamase in a conjugative plasmid.

Enterobacter cloacae strains are still infrequently resistant to carbapenems. A carbapenem-resistant clinical isolate of E. cloacae producing a plasmid-mediated metallo-beta-lactamase (MBL), VIM-4, was recovered from a Greek hospitalized patient. The bla(VIM-4) gene was located in the variable array of a class 1 integron structure repeatedly detected among bla(VIM-1)-bearing Gram-negative pathogens in Greece. The isolate possessed also on the same conjugative plasmid an extended-spectrum beta-lactamase (ESBL), SHV-2a, which contributed to the beta-lactam-resistant phenotype. This is the first report showing co-transfer of an ESBL with a VIM-type MBL. It suggests also that different VIM-type gene cassettes have been incorporated in a common integron structure, which seems indigenous of Gram-negative pathogens in our region.

A rare case of Brucella melitensis infection in an obstetrician during the delivery of a transplacentally infected infant.

We report a case of Brucella melitensis infection in an obstetrician who was infected during the delivery of an infant suffering from congenital brucellosis. The obstetrician was treated with doxycycline and rifampin and fully recovered. This is the first reported case of Brucella infection transmitted through infectious secretions at the delivery of a transplacentally infected newborn and emphasizes that, especially in endemic areas educational and technical measures are needed in order the obstetricians to avoid ingestion of secretions during clearance of the newborn's respiratory tract from saliva and amniotic fluid.

Phenotypic assessment of Neisseria meningitidis isolates obtained from patients with invasive meningococcal disease in Greece, 1993-2003: implications for serogroup B vaccines based on PorA serosubtype antigens.

Serogroup B is the major isolate from patients with invasive meningococcal disease (IMD) in Greece. This study used the whole cell enzyme-linked immuosorbent assay (ELISA) with monoclonal antibodies to screen Neisseria meningitidis isolates obtained from patients with IMD between 1993 and 2003 to determine if serosubtypes included in the hexavalent Por A OMP vaccines being tested in northern Europe were prevalent in Greece. During this period there were significant changes in the proportions of serogroups B and C isolated from patients. Serogroup C was predominant in 1996-1997 but fell sharply with corresponding increases in serogroup B. Of the 591 isolates sent to the National Meningitis Reference Laboratory in Athens during this period, 325 (55%) were serogroup B. Among those tested for serosubtype, porA proteins used for the vaccine being tested in Britain were detected on 85/284 (30%) strains and for the vaccine being tested in the Netherlands 175/284 (62%). P1.14 (58/284, 20%) the predominant serosubtype among the Greek isolates, is not present in either vaccine formulation; 23/284 (8%) strains did not react with any of the monoclonal antibodies. Our results indicate that introduction of the vaccines currently being evaluated in northern Europe would not be warranted in the Greek population.

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece.

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.