Juan Carlos Sanz

Clonal Spread of Levofloxacin-Resistant Streptococcus pneumoniae Invasive Isolates in Madrid, Spain, 2007 to 2009

ABSTRACT Among 1,349 Streptococcus pneumoniae invasive isolates, 45 (3.3%) were levofloxacin resistant. Serotype distribution was as follows: 8 (n = 32 isolates), 19A (n = 4 isolates), 7F (n = 3 isolates), 9V (n = 2 isolates), 10A (n = 1 isolate), 19F (n = 1 isolate), 6B (n = 1 isolate), and nontypeable (n = 1 isolate). Levofloxacin-resistant isolates had dual mutations in the gyrA and parC genes. Serotype 8 strains corresponded to a capsular switching of the Sweden15A-25 clone. Levofloxacin resistance was also detected among multiresistant (ST27619A, Spain9V-ST156, ST8819F, and ST15426B) and among usually antibiotic-susceptible (Netherlands7F-ST191, ST120119A, and ST263910A) clones.

Multidrug‐resistant pneumococci (serotype 8) causing invasive disease in HIV+ patients

From July 2007 to June 2009, all pneumococci causing invasive pneumococcal disease in our hospital were serotyped. Antimicrobial susceptibility was determined by microdilution. Molecular typing was performed by pulsed-field gel electrophoresis and by multilocus sequence typing. Among 251 invasive pneumococci, serotype 8 was the most frequent (13.5%). All serotype 8 strains were susceptible to penicillin; however, 61.8% (21/34) were co-resistant to erythromycin, levofloxacin and tetracycline and identical to the Sweden(15A) -ST63 clone. Serotype 8 was significantly more frequent among human immunodeficiency virus (HIV)-infected patients (36.5%). The high prevalence of this non-conjugate vaccine multiresistant serotype 8 is a cause for concern mainly in HIV-infected patients.

Mumps virus diagnosis and genotyping using a novel single RT-PCR

BACKGROUND IgM detection is considered as the gold standard for mumps diagnosis. Currently, most cases in developed countries occur in highly vaccinated populations due to secondary vaccine failure. In these patients, pre-existing vaccine-induced antibodies are not able to neutralise the virus, but prevent the typical primary response, so that specific IgM is not always elicited. Consequently, acute infection has to be demonstrated by direct detection of the virus by viral isolation or genomic amplification. RT-PCR allows a diagnosis with the maximum sensitivity to be made and also forms the basis for genotype characterisation by sequencing the SH gene, according to WHO recommendations. However, none of the RT-PCR techniques properly evaluated for the diagnosis of acute mumps infection yields an amplification fragment useful for genotyping, and none of the amplification techniques described for genotyping has proved to be sensitive enough for diagnosis. OBJECTIVES Development of a RT-PCR for the mumps virus diagnosis and genotyping, properly evaluated in comparison with serological gold-standard technique. STUDY DESIGN 195 suspected mumps cases and six wild type MuV genotypes were studied. RESULTS Our method was able to detect 0.001 TCID(50) of mumps virus. Fifty-eight of these showed positive results, of which 54 (93.3%) showed mumps RNA in saliva, while only 20 (34.5%) had mumps IgM in serum. Genotypes G1, G2, H1, H2, D1 and C were identified in positive samples. CONCLUSIONS The technique described could be a very useful tool for mumps surveillance, management and control.

Comparison of chemiluminescent immunoassay and ELISA for measles IgG and IgM

In the context of measles elimination, the identification of recent infections is important for clinical laboratories. Serological diagnosis is achieved by detecting specific IgG and IgM. Recently an automated chemiluminescent immunoassay (CLIA) (Liaison, DiaSorin, Italy) has been used to quantify the measles antibody. The aim of this study was to compare this assay with Enzygnost ELISA (Siemens, Germany), with final classification of discrepancies by indirect immunofluorescence (Euroimmun, Germany). For measles IgM, 204 sera were analyzed: 50 IgM‐positive, 104 IgM‐negative/IgG‐positive, and 50 from other viral infections (B19V, rubella, mumps, CMV, and EBV). For the measles IgG assay, 162 samples were tested: 106 were positive and 56 were negative. For measles IgM, the sensitivity and specificity of CLIA against ELISA were 94% (95% CI: 83.2–98.6) and 100% (95% CI: 97.1–100), respectively; the corrected figures after the final classification of discrepancies were 100% (95% CI: 91.0–100) and 99.4% (95% CI: 96.1–100), respectively. In relation to IgG, the sensitivity and specificity of CLIA against ELISA were, respectively, 97.2% (95% CI: 91.7–99.4) and 92.9% (95% CI: 82.5–97.7), and 95.5% (95% CI: 89.5–98.3) and 100% (95% CI: 91.8–100) after the final classification. CLIA showed excellent sensitivity and specificity in detecting measles IgG and IgM antibodies, eliminating the need to aliquot specimens before carrying out the assay.

Application of a Commercial Immunoblot to Define EBV IgG Seroprofiles

Immunoblot (IB) techniques using different Epstein–Barr virus (EBV) antigens have been applied for detecting specific antibodies, making possible to obtain EBV seroprofiles in a single determination. The aim of this study was to evaluate a commercial IB for the detection of EBV‐specific IgG (Euroimmun, Lübeck, Germany).

Identification of Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid by multiplex real-time PCR

INTRODUCTION The aim was to evaluate the utility of a multiplex real-time PCR to detect Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid (PF). METHODS A collection of 81 PF samples was used. Sixty were considered positive for S. pneumoniae according to previous results (54 by an in-house lytA gene PCR and eight by universal rRNA PCR). RESULTS The sensitivity for detection of the lytA, plyA and psaA genes by multiplex PCR was 100% (60/60), 98.3% (59/60) and 91.7% (55/60), respectively. The detection of all three genes was negative in 21 samples formerly confirmed as negative for S. pneumoniae (100% specificity) by the other procedures (9 by in-house lytA PCR and 12 by rRNA PCR). CONCLUSIONS The use of this multiplex PCR may be a useful option to identify S. pneumoniae directly in PF samples.

Comparison of commercial methods of immunoblot, ELISA, and chemiluminescent immunoassay for detecting type-specific herpes simplex viruses-1 and -2 IgG

Serology for type‐specific herpes simplex virus (HSV) is based on the use of the respective glycoprotein G (gG).

AP-PCR as Typing Method in Clinical Isolates of Helicobacter pylori

H. pylori is accepted as the most important cause of human chronic active gastritis and is also associated with duodenal ulcer. Its clearance from the gastric mucosa cures ulceration but, relapse following treatment is often associated with the isolation of H. pylori from a biopsy sample.

Evolution of the Resistance to Several Antibiotics in Helicobacter pylori over a Four-Year Period

A variety of antimicrobial agents display good activity against H. pylori in vitro, but when using as single agent in clinical studies, they do not succeed in eradicating the organism. Several regimes have been proposed in the treatment of the pathology produced by H. pylori with at least 2 or 3 drugs, including one or two antibiotics and other compounds such as bismuth salts and proton pump inhibitors as omeprazole, lansoprazole and pantoprazole, that have shown activity against the microorganism with minimal inhibitory concentrations (MICs) of 16–64 mg/l9. Amoxicillin, tetracycline, metronidazole and clarithromycin are the most frequent antimicrobial agents used for the treatment of H. pylori infection.