False-positive of blood culture instrument: leukocytosis, overfilled vials or defective position?

Continuous monitoring of the performances of blood culture instrument can be based on the indicators proposed by the QUAMIC. Of these, the analytic performance indicator evaluates the rate of false-positive vials. False-positives vials can be reported by the device in case of leukocytosis or when vials are overfilled. In our laboratory, we record prospectively in our software all false positive vials as well as the position used for their incubation. These data are analyzed at least twice a year. For the first half of 2016, this strategy allowed us to identify a defective position. We then evaluated the impact of this anomaly as very weak. The one-year follow-up after the position repair confirmed a correction of the problem. Thus, traceability of positions reporting unexplained false-positive vials (i.e. neither due to leukocytosis nor overfilled vials) can allow laboratories to identify a defective position. This survey, if done prospectively, is simple to perform and not time-consuming. It could usefully complement the analytical performance indicator based on the false-positive rate.

Chromosome-Encoded Broad-Spectrum Ambler Class A ß-Lactamase RUB-1 from Serratia rubidaea.

Whole-genome sequencing of Serratia rubidaea CIP 103234T revealed a chromosomally located Ambler class A ß-lactamase gene. The gene was cloned, and the ß-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter ß-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ70 consensus sequence. This work further illustrates the heterogeneity of ß-lactamases among Serratia spp.

[Investigation of hepatitis B and hepatitis C virus infections by serological and molecular methods in hemodialysis patients]. / Hemodiyaliz hastalarinda hepatit B ve hepatit C virus enfeksiyonlarinin serolojik ve moleküler yöntemlerle arastirilmasi

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are significant causes of morbidity and mortality in hemodialysis patients, since those patients are highly susceptible to infections due to immune suppression. The aims of this study were to investigate the presence of HBV and HCV infections in chronic hemodialysis patients by serological and molecular methods, and to determine the rate of occult HBV infection and the viral genotypes. A total of 201 patients who were under hemodialysis due to end-stage renal disease, were retrospectively evaluated. The study involved the patients at three different centers in Antalya, Turkey during 2006. HBV and HCV markers in the patients' sera were screened by ELISA method, viral nucleic acids were investigated by real-time polymerase chain reaction (PCR) in patients' plasma and viral genotypes were determined by DNA sequence analysis. Detection of at least one of the HBV markers HBsAg, anti-HBc total, and HBV DNA, was accepted as HBV infection, and detection of anti-HCV and/or HCV RNA was accepted as HCV infection. HBsAg positive patients with negative HBV DNA were considered as occult HBV infection. Of the patients 80 (40%) were female, 121 (60%) were male and the mean age was 51.16 ± 16.28 (range 17-93) years. In our study, sole anti-HBs positivity due to HBV vaccination, was detected in 89 (44.3%) patients. One hundred (50%) patients were found positive in terms of HBV infection and 40 (20%) were positive for HCV infection, while 24 (12%) patients had HBV and HCV co-infections. Eighty-five (42.3%) patients had no HBV and HCV infection. Among the 5 (2.5%) patients who were HBsAg positive, four were also HBV DNA positive. Occult HBV infection was detected in 1 (0.5%) patient. Anti-HCV and HCV RNA were found positive in 37 (18.4%) and in 24 (12%) patients, respectively. Among the HCV-RNA positive patients, 3 (12.5%) were anti-HCV negative. ALT and AST levels were found normal in all of the HBV DNA positive patients, and 62.5% (15/24) of HCV RNA positive patients. All of the HBV isolates were identified as genotype D and HCV isolates as genotype 1b. No statistically significant correlation was detected between the HBV infection and patients' age, duration of hemodialysis and elevation of serum transaminase levels (p> 0.05). On the other hand, HCV infection was seen to increase with age (p= 0.047). HCV infection showed a statistically significant increase with the duration of hemodialysis. HCV infection risk was increased in patients who were under hemodialysis for ≥ 25 months (p< 0.001, OR: 0224, 95% CI= 0089-0562). There was also a statistically significant correlation between the presence of HCV infection (anti-HCV and/or HCV RNA positive) and high levels of serum transaminases (p< 0.001). However, in two of the three cases who were anti-HCV negative and HCV RNA positive, serum transaminase levels were normal while the viral loads were high. Therefore to follow-up HCV infection in the hemodialysis patients, anti-HCV and serum transaminase levels may not be sufficient alone and these patients should be evaluated periodically for HCV RNA. In addition, the detection of occult HBV infection in one of the study patients, indicated that HBV DNA should also be investigated at regular intervals in the hemodialysis patients.

Real-time PCR for detection of blaOXA-48 genes from stools.

OBJECTIVES: Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range. METHODS: An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants. RESULTS: qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other ß-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum ß-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity. CONCLUSIONS: The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

Structural variability of the herpes simplex virus 1 genome in vitro and in vivo.

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation. In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the one in vitro was observed, along with a considerable proportion of noncanonical assortment.

Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples.

An in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification of bla(NDM-1) DNA was linear over 10 log dilutions (r(2) = 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other ß-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 10(1) to 3 × 10(1) CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 4 × 10(3) CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 10(1) CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the bla(NDM-1) qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time for bla(NDM-1) detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

Meganuclease-mediated Inhibition of HSV1 Infection in Cultured Cells.

Herpes simplex virus type 1 (HSV1) is a major health problem. As for most viral diseases, current antiviral treatments are based on the inhibition of viral replication once it has already started. As a consequence, they impair neither the viral cycle at its early stages nor the latent form of the virus, and thus cannot be considered as real preventive treatments. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as meganucleases. With the aim of a proof of concept study, we generated several meganucleases recognizing HSV1 sequences, and assessed their antiviral activity in cultured cells. We demonstrate that expression of these proteins in African green monkey kidney fibroblast (COS-7) and BSR cells inhibits infection by HSV1, at low and moderate multiplicities of infection (MOIs), inducing a significant reduction of the viral load. Furthermore, the remaining viral genomes display a high rate of mutation (up to 16%) at the meganuclease cleavage site, consistent with a mechanism of action based on the cleavage of the viral genome. This specific mechanism of action qualifies meganucleases as an alternative class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms.

[Investigation of Panton-Valentine leukocidin gene, SCCmec gene cassette types and genotypes of methicillin-resistant Staphylococcus aureus strains isolated from outpatients]. / Ayaktan saglik hizmeti alan hastalardan izole edilen metisiline dirençli Staphylococcus aureus izolatlarinda Panton-Valentin lökosidin geni ve SCCmec gen kaseti tiplerinin arastirilmasi ve izolatlarin genotiplendirilmesi.

The identification of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) is becoming a hard task since colonization with MRSA is lasting for years and the number of the health care facilities other than hospitals is continuously increasing. In this study we aimed to investigate the genetic properties and health-care association of MRSA strains isolated from skin and soft tissue infections of outpatients admitted to Akdeniz University Hospital. Thirty strains were phenotypically identified as MRSA and after assessing the risk factors, 28 (93.3%) of them were classified as health-care associated (HCA) and 2 (6.7%) of them as community-acquired (CA). All of the isolates were positive for nuc and mecA genes by polymerase chain reaction. Antimicrobial resistance rates of HCA-MRSA and CA-MRSA isolates were found as follows, respectively; 89.3% and 0% for rifampin, 89.3% and 50% for ciprofloxacin, 89.3% and 0% for gentamicin, 50% and 50% for erythromycin, 28.6% and 0% for clindamycin, whereas all of the isolates were susceptible to vancomycin, linezolid and trimethoprim-sulfamethoxazole. SCCmec type III was detected in 24 (85.7%) of HCA-MRSA strains. SCCmec type IV was detected in 1 (3.6%) of HCA-MRSA and in 2 (100%) of CA-MRSA strains. Panton-Valentin leucocidin (PVL) gene positivity was detected in only CA-MRSA isolates (2/2; 100%). MRSA isolates were grouped into 17 different genotypes (from A to R) of which pulsotype A was predominant among HCA isolates and CA-MRSA isolates were found to be clonally related with each other. This is the first study which investigated the genetic properties of MRSA strains in Antalya (a province located at Mediterranean Region, Turkey). In this study HCA risk factors were investigated and CA-MRSA rate was only 6.7% among all MRSA strains isolated from outpatients. As a result of detailed investigation of HCA risk factors, it was possible to detect the exact rate of CA-MRSA among outpatients. Thus it is of clinical and epidemiological importance to know the origin of MRSA isolates since this will affect the empirical treatment choice. Genetic studies supplied by appropriate demographic data will help to clarify the evolution and epidemiology of MRSA in the community and in the hospital setting.

Rapid detection of CTX-M-producing Enterobacteriaceae in urine samples.

OBJECTIVES: CTX-M extended-spectrum beta-lactamases (ESBLs) are emerging worldwide. Fast and reliable detection techniques may become mandatory for implementing proper treatment and infection control measures. Here, a bla(CTX-M)-specific LightCycler real-time PCR (LC-PCR) assay based on hybridization probes was developed. METHODS: Urine samples positive for Gram-negative bacilli as revealed by Gram staining were collected over a 3 month period at Bicêtre hospital, France. Aliquots of these urine samples were frozen for subsequent molecular analysis, and the bacteria were cultured and identified by standard bacteriological techniques (biochemical tests, disc diffusion antibiogram and synergy testing). LC-PCR and standard PCR followed by sequencing was performed on all ESBL-positive and on 70 randomly chosen ESBL-negative urine samples. RESULTS: Over the study period, 810 urine samples were collected from 655 patients. Thirty-six ESBL-producing Enterobacteriaceae, mostly Escherichia coli (77%), were identified from 29 patients, of which half were outpatients. Twenty-five urine samples (19 patients) were found to be positive for bla(CTX-M) genes using the LC-PCR assay. The bla(CTX-M) genes belonged to the bla(CTX-M-1), bla(CTX-M-9) and bla(CTX-M-2) groups (68%, 24% and 8%, respectively). Standard PCR and sequencing of the entire bla(CTX-M) genes confirmed the LC-PCR results; 17 CTX-M-15, 6 CTX-M-9 and 2 CTX-M-2. Among the remaining ESBLs, eight were of the TEM type and three of the SHV type. CONCLUSIONS: The LC-PCR assay represents a powerful tool for rapid identification of CTX-M producers in urine samples.