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.

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.