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Contains fulltext : 136460.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. METHODOLOGY: A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. RESULTS: The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. CONCLUSIONS: The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.

Background: VIM-type metallo-betalactamases (MBLs) exhibit hydrolytic activity against most betalactam antibiotics, including carbapenems. So far, VIM-type-producing Klebsiella pneumoniae isolates had not been reported in Latin America. Methodology: In July 2005, a carbapenem-resistant Klebsiella pneumoniae was isolated from a urine sample collected from a 7-year-old girl hospitalized at the Hospital de Ninos "J. M. de los Rios" in Caracas, Venezuela. This strain was identified using conventional biochemical tests. The susceptibility analysis was conducted by disk diffusion, and MICs for Imipenem and Meropenem were performed by agar dilution. For the phenotypic detection of MBL we used the Imipenem-EDTA/SMA double-disk diffusion method. The hydrolytic activity against carbapenems was determined by the Masuda microbiological method. Purified protein was subjected to isoelectric focusing (IEF). Detection of antimicrobial resistance genes was performed by PCR amplification with specific VIM primers. Results: The strain showed resistance to most betalactam antibiotics, quinolones and amynoglicosides, but remained susceptible to Aztreonam and Cefepime. The use of phenotypic and microbiological methods detected the presence of a metallobetalactamase. By IEF we visualized three bands at pI 5.4, 7.6 and 7.9, corresponding to reduced-spectrum betalactamases, and a band at pI 5.8 that corresponded to the metallobetalactamase. PCR screening of bla genes revealed the presence of bla VIM , with an amplicon of 261 bp. Conclusions: This is the first report of a MBL-mediated carbapenem-resistant Klebsiella pneumoniae in Latin America, which constitutes a public health concern in our region since their transference to other microorganisms with multiple antibiotic resistance mechanisms will increase the antimicrobial resistance problem.