Evaluation of an automated extraction method for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Cobas Amplicor PCR from different sample materials

A commercially available automated device (MagNA Pure LC) was adapted for nucleic acid extraction of urogenital specimen for subsequent PCR detection of Chlamydia trachomatis and Neisseria gonorrhoeae in a clinical laboratory. Results were compared to the standard manual extraction procedure and showed excellent correlation, with even slightly increased sensitivity.     

Multiplex Strategy for Multilocus Sequence Typing,fla Typing,and Genetic Determination of Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolates Collected in Switzerland

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost- and time-efficient routine monitoring.Infection with Campylobacter has become the major cause of bacterial enteritis in Europe and other parts of the developed world, overtaking Salmonella infection (8). Campylobacter jejuni accounts for approximately 90% of all Campylobacter infection cases, whereas C. coli is responsible for approximately 10% of infections. Other Campylobacter species, such as C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus, are sporadically found (24). Due to the fact that Campylobacter is mostly commensal in the enteron of many warm-blooded animals used for meat production, campylobacteriosis is a zoonotic disease. Quality control, monitoring, and eventually tracing of contaminated food products is therefore important for public health reasons. Campylobacter typing by applying various, mostly genetic, methods is used for this purpose. Classical pulsed-field gel electrophoresis and amplified fragment length polymorphism, as well as flaA typing based on the restriction analysis of PCR-amplified fragments or sequencing of the flagellin-encoding gene, have been described for Campylobacter (20, 37). Recently, multilocus sequence typing (MLST) has been established as a highly reproducible method allowing precise and simple worldwide comparison of types, and it is becoming the gold standard in this field (4-6,13,17-19, 22, 23, 30, 33). Despite its many advantages, MLST is still time-consuming and expensive and therefore not feasible for routine testing. For example, the scheme for C. jejuni typing recommended by the PubMLST database hosted by the University of Oxford, Oxford, United Kingdom (http://pubmlst.org/campylobacter) includes a total of 51 different primers to be used for PCR amplification and sequencing of the seven target gene sequences. Another 14 primers are described for MLST of C. coli. With problematic isolates, optimal primer combinations have to be determined, and reactions have to be repeated in order to obtain all seven allele sequences needed for sequence type (ST) determination.MLST alone provides excellent information about the global epidemiology and population structure of Campylobacter, but it appears to be less discriminative in short-term epidemiological studies (28). The addition of more variable targets, such as flagellin-encoding genes, increases the discriminatory power of sequence-based typing. The most frequently used gene for this purpose is flaA (2, 5, 7, 17, 20, 26, 29), although flaB is also used, and as a more stable gene, flaB might become more important (21). Other important factors to consider are the time and effort needed to perform the appropriate data analysis, especially in the context of internationally standardized approaches and the use of publicly available typing tools, such as http://pubmlst.org.Since the 1990s, the prevalence of antibiotic resistance has increased dramatically in both animal and human Campylobacter isolates. This is especially the case for quinolone resistance, the emergence of which is correlated with the introduction of quinolones in the treatment of food-producing animals. The emergence of macrolide-resistant Campylobacter isolates has also been observed but until recently was less pronounced than quinolone resistance (41). Quinolone resistance is mainly based on a point mutation in the gyrase gene, gyrA (C257T or, less frequently, A256G) (1). In the case of macrolide resistance, it is caused by a point mutation (A2075G or A2074C) in the loop in domain V of the 23S rRNA gene (34).In order to optimize and simplify the amplification and sequencing strategy for MLST and combine it with sequence-based fla typing, as well as with antibiotic resistance determination, we established a modular and adaptable multiplex PCR and sequencing protocol using the minimum number of primers, which can be used equally well for C. jejuni and C. coli. About 95% of human Campylobacter infections can be covered with our typing scheme. Proper identification of Campylobacter isolates is not always trivial, and misidentification might hamper downstream typing, especially genotyping. 16S rRNA and rpoB genes were included in the multiplex approach as a basic genetic identification module for the genus Campylobacter, and the discriminatory power at the species level was examined. Through this approach, enteritis-causing Campylobacter species other than C. jejuni and C. coli are dealt with by proper identification.The robustness of the multiplex approach was tested on more than 300 C. jejuni and C. coli strains. Data analysis was performed using a newly developed Internet-based Integrated Database Network System (IDNS) (SmartGene, Zug, Switzerland) platform for genotyping Campylobacter.     

Bloodstream infections caused by small-colony variants of coagulase-negative staphylococci following pacemaker implantation.

Small-colony variants (SCVs) of Staphylococcus aureus cause persistent and relapsing infections. Relatively little is known regarding infections caused by SCVs of coagulase-negative staphylococci. We report two cases of pacemaker electrode infections due to SCVs of Staphylococcus epidermidis and Staphylococcus capitis. Sequence analysis of a portion of the 16S rRNA gene (16S rDNA) confirmed the identity of the staphylococcal species as S. capitis and S. epidermidis. Isolates from cultures of blood obtained over at least a 2-week interval were compared by pulsed-field gel electrophoresis and found to be clonal even though the colony morphology was very different. Analysis for auxotrophism revealed hemin dependencies for all isolated SCVs. The two cases have several clinical and laboratory characteristics (which are also seen with S. aureus SCV infections) and strongly suggest that SCVs of coagulase-negative staphylococci must be actively sought, because they grow very slowly and can be easily missed.     

Interaction of insulin-like growth factor II with rat chondrocytes: receptor binding, internalization, and degradation

We have characterized the interaction of insulin-like growth factor II (IGF-II) with its plasma membrane receptor(s) on cultured rat chondrocytes. Our studies, paralleling those already reported for IGF-I, demonstrate that [125I]IGF-II binds to these receptors with a high degree of affinity and that this process is reversible, specific, and time, temperature, and concentration dependent. At 4 C, unlabeled IGF-II causes half-maximal displacement of the labeled ligand at a concentration of 22 ng/ml, whereas IGF-I is approximately 1/200th as potent, and insulin does not displace [125I]IGF-II even at a concentration of 10 micrograms/ml. Maximum binding to chondrocytes (44% of added radioactivity) occurred after 4-5 h of incubation at 15 C. Compared to [125I]IGF-I binding, this value is 7-fold higher and is consistent with an affinity constant (Ka = 3.8 X 10(8) M-1) approximately 1 order of magnitude greater. Photoaffinity labeling studies disclose that IGF-II binds primarily to the type II IGF receptor, which has an apparent mol wt of 220K when electrophoresed under nonreducing conditions and 270K under reducing conditions. Nanomolar concentrations of IGF-II stimulated the synthesis of DNA and RNA in a dose-related manner, and micromolar concentrations of insulin demonstrated an additive effect with respect to the incorporation of [3H]thymidine into DNA, but not [3H]uridine into RNA. Preincubation of rat chondrocytes with increasing concentrations of insulin caused a marked dose-related increase in [125I]IGF-II binding, a phenomenon previously reported in several other cell types. In addition to defining the binding characteristics of IGF-II, we used the lysosomotropic agents chloroquine and ammonium chloride to demonstrate that its ligand-receptor complex, like that of IGF-I, is internalized and degraded partially via the lysosomal pathway.     

Rationality in medical decision making: a review of the literature on doctors' decision-making biases

The objectives of this study were to describe ways in which doctors make suboptimal diagnostic and treatment decisions, and to discuss possible means of alleviating those biases, using a review of past studies from the psychological and medical decision-making literatures. A number of biases can affect the ways in which doctors gather and use evidence in making diagnoses. Biases also exist in how doctors make treatment decisions once a definitive diagnosis has been made. These biases are not peculiar to the medical domain but, rather, are manifestations of suboptimal reasoning to which people are susceptible in general. None the less, they can have potentially grave consequences in medical settings, such as erroneous diagnosis or patient mismanagement. No surefire methods exist for eliminating biases in medical decision making, but there is some evidence that the adoption of an evidence-based medicine approach or the incorporation of formal decision analytic tools can improve the quality of doctors' reasoning. Doctors' reasoning is vulnerable to a number of biases that can lead to errors in diagnosis and treatment, but there are positive signs that means for alleviating some of these biases are available.     

Mycobacterium heckeshornense sp. nov., A new pathogenic slowly growing Mycobacterium sp. Causing cavitary lung disease in an immunocompetent patient

A pathogenic scotochromogenic Mycobacterium xenopi-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making two surgical interventions necessary after years of chronic disease. This case prompted us to characterize this mycobacterium by a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers which were typical for the genus Mycobacterium, i.e., the meso isomer of 2,6-diaminopimelic acid, arabinose, and galactose as diagnostic whole-cell sugars, MK-9(H(2)) as the principal isoprenoid quinone, a mycolic acid pattern of alpha-mycolates, ketomycolates, and wax ester mycolates, unbranched saturated and unsaturated fatty acids plus a significant amount of tuberculostearic acid, and small amounts of a C(20:0) secondary alcohol. On the basis of its unique 16S rRNA and 16S-23S spacer gene sequences, we propose that the isolate should be assigned to a new species, Mycobacterium heckeshornense. This novel species is phylogenetically closely related to M. xenopi. The type strain of M. heckeshornense is strain S369 (DSM 44428(T)). The GenBank accession number of the 16S rRNA gene of M. heckeshornense is AF174290.     

Two different 16S rRNA genes in a mycobacterial strain.

Sequencing of the gene coding for 16S rRNA (16S rDNA) is a well-established method used to identify bacteria, particularly mycobacteria. Unique sequences allow identification of a particular genus and species. If more than one 16S rDNA is present on one mycobacterial genome, their sequences are assumed to be strictly or almost identical. We have isolated a slowly growing Mycobacterium strain, "X", identified by conventional biochemical tests as Mycobacterium terrae. Identification by amplification and direct sequencing of 16S rDNA yielded ambiguous results in two variable regions, suggesting the presence of different copies of the sequenced gene. Total DNA was digested by restriction enzymes and hybridized after Southern blotting to a probe representing about two-thirds of the 16S rDNA. Two copies of 16S rDNA were identified and cloned. By sequencing, the clones were of two different types, A and B, differing in 18 positions. Oligonucleotides specific to each copy of the 16S rDNA were used to distinguish the positions of the two genes observed in the Southern blot. We conclude that Mycobacterium strain "X" has two different copies of 16S rDNA. Variations in the sequence between two copies of 16S rDNA gene have been described in archaeobacteria, but not in mycobacteria. When placed in a phylogenetic tree together with other slowly growing mycobacteria gene A shows a common root with M. terrae, whereas gene B is placed separately.