[Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis]. / Francisella tularensis'in moleküler tanisinda yeni gelistirilen kullanima hazir ticari PCR kitinin etkinliginin degerlendirilmesi.

Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR Toolbox assay were found to be 100%. The lowest detection limit of this method was determined as 100 genomic equivalent per PCR reaction mix. The new PCR kit is a rapid and accurate alternative to the conventional PCR methods since the toolbox includes all of the required chemicals, accessories and equipments. This ready-to-use PCR assay was appraised to be a valuable diagnostic tool for the detection of F.tularensis in the outbreak settings particularly in remote areas with limited resources.

Nasopharyngeal carriage of Streptococcus pneumoniae in healthy Turkish children after the addition of PCV7 to the national vaccine schedule.

UNLABELLED: The aim of this study was to determine serotype distribution and investigate antimicrobial resistance patterns of Streptococcus pneumoniae in healthy Turkish children in the era of community-wide pneumococcal conjugate vaccine (PCV7). The study was conducted on 1,101 healthy children less than 18 years of age. Specimens were collected with nasopharyngeal swabs between April 2011 and June 2011. Penicillin and ceftriaxone susceptibilities were determined by E-test according to the 2008 Clinical Laboratory Standards Institute, and serotypes of the isolates were determined by Quellung reaction. The nasopharyngeal pneumococcal carriage rate was 21.9 % (241/1,101). Using the meningitis criteria of minimum inhibitory concentration values, 73 % of the isolates were resistant to penicillin and 47.7 % of them were resistant to ceftriaxone. Half of all pneumococcal isolates were serotyped as 19F (15.2 %), 6A (15.2 %), 23F (10.3 %), and 6B (9.3 %) and surprisingly, no serotype 19A was isolated. Serotype coverage rates of PCV7 and non-PCV7 were 46.2 and 53.8 %, respectively. The most common penicillin- and ceftriaxone-resistant serotypes were 6A, 6B, 14, 19F, and 23F. Penicillin- and ceftriaxone-resistant isolates were more prevalent in serotypes covered by PCV7 than the non-PCV7 serotypes. CONCLUSION: After the community-wide PCV7 vaccination, more non-PCV7 serotypes were isolated from the carriers compared to the time before PCV7 was used especially the serotype 6A, and the antimicrobial resistance of pneumococci was significantly increased.

In vitro susceptibility of isolates of Francisella tularensis from Turkey.

BACKGROUND: Tularemia is an infection caused by Francisella tularensis, which has a wide distribution in the northern hemisphere and diverse clinical manifestations. For decades, the drug of choice for the treatment of tularemia has been streptomycin, with tetracycline and chloramphenicol being used as alternatives. The purpose of the present study was to determine the in vitro antimicrobial susceptibility of a large panel of geographically diverse F. tularensis isolates from Turkey against traditional and newer antimicrobial agents. METHODS: The antibiotic susceptibilities of 250 F. tularensis strains were examined using the Epsilometer test for 9 antimicrobial agents. Each isolate was identified by conventional and molecular techniques. RESULTS: All the strains were confirmed biochemically and using a combination of species- and subspecies-specific polymerase chain reaction (PCR) assays to be F. tularensis subsp. holarctica. One isolate was assigned to F. tularensis subsp. holarctica biovar japonica based on erythromycin susceptibility, an ability to ferment glycerol, and the nucleotide sequence of the region of difference 1 (RD1). All strains were susceptible to aminoglycosides (streptomycin and gentamicin), tetracyclines (tetracycline and doxycycline), chloramphenicol, 2 fluoroquinolones (ciprofloxacin and levofloxacin), and rifampicin. In addition, all isolates except 1 had a minimal inhibitory concentration (MIC) for erythromycin of > 256 µg/ml. CONCLUSIONS: Since the fluoroquinolones showed the lowest MIC values and have advantages such as excellent bioavailability and activity, availability of oral formulations, and lower toxicities, they represent candidate therapeutic options in the first-line treatment of tularemia. To the best of our knowledge, this is the first report of the presence of F. tularensis subsp. holarctica biovar japonica outside Japan.

Risk factors for nasopharyngeal carriage of Streptococcus pneumoniae in healthy Turkish children after the addition of heptavalent pneumococcal conjugate vaccine (PCV7) to the national vaccine schedule.

The purpose of this study was to investigate the effects of pneumococcal conjugate vaccine (PCV7) on nasopharyngeal (NP) carriage rates of Streptococcus pneumoniae in healthy Turkish children. The study was conducted on 1101 healthy Turkish children between 1 month and 18 years of age. The median and mean ages of the children were 25 months (1 month-18 years) and 45.7±49.6 months, respectively. S. pneumoniae was isolated in 241/1101 (21.9%) children included in the study. According to multivariate analysis, being <5 years of age, presence of a child attending a daycare center, recovery from respiratory infection within the last month, low income level of the family, and presence of more children in the family were found to be the risk factors for the NP pneumococcal carriage. The carriage rate of NP pneumococci in healthy children was not influenced by PCV7 in Turkey.