An assessment of pipette calibration stability using statistical process control charts

Routine pipette calibration is an essential part of any quality assurance and quality control program in the forensic sciences and beyond. Pipette calibration standards in a forensic laboratory are typically set to the limits outlined by the document ISO8655, published by the International Organization for Standardization for the general scientific community. Alternative methods exist that may be capable of monitoring pipette stability across time in a forensic setting. Statistical process control charts, or Shewhart charts, are one such form of process control, which is being investigated for its potential application to pipette calibration monitoring for forensic DNA laboratories. Indeed, the application of process control lines for monitoring the calibration of volumetric equipment is not without precedent.1 To investigate the applicability of process control charts for monitoring pipette stability, a series of X ̅ and S charts, a type of Shewhart chart, have been produced from eight years of collected calibration data. A total of 71 pipettes of the following sizes were examined: 1-10 µL, 1-10 µL multi-channel, 10-100 µL, 100-1000 µL, 1-3 µL, 30-300 µL, 5-50 µL, 5-50 µL multi-channel, and 500-5000 µL pipettes. The ISO8655 calibration recommended volume limits of these pipettes have been added to the charts for the purposes of comparison. With these charts, it is possible to assess pipette performance over time in comparison to the ISO8655 calibration standards and to the control limits imposed by the Shewhart charts. The completed charts suggest that the methodology proposed by Shewhart shows promise as a supplement to ISO8655 recommendations for monitoring pipette stability across time. To corroborate the value of using Shewhart charts to monitor pipette performance, a serial dilution study in conjunction with a series of simulations with dynamic modeling software was performed. This dilution study investigated whether the systematic biases shown by the Shewhart charts could be measured in a laboratory setting. The simulations investigated multiple hypothetical pipetting scenarios concerning various levels of systematic bias. The simulations consistently corroborated the value of Shewhart charts to enforce better compliance between a pipette’s nominal and actual volume delivery, while the serial dilution study offered partial evidence of systematic pipetting bias

Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing

<p>Abstract</p> <p>Background</p> <p><it>Bacillus cereus </it>is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype <it>B. cereus </it>and analysis has suggested a clonal or weakly clonal population structure for <it>B. cereus </it>and its close relatives <it>B. anthracis </it>and <it>B. thuringiensis</it>. In this study we used MLST to determine if <it>B. cereus </it>isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.</p> <p>Results</p> <p>A retrospective analysis of 55 clinical <it>B. cereus </it>isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27), gastrointestinal (GI) illness (n = 18), and associated isolates from food (n = 10) were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST) in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates.</p> <p>Conclusion</p> <p>STs of clinical <it>B. cereus </it>isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to <it>B. anthracis </it>and emetic <it>B. cereus </it>isolates.</p

Chronic typhoid infection and the risk of biliary tract cancer and stones in Shanghai, China

Previous studies have shown a positive association between chronic typhoid carriage and biliary cancers. We compared serum Salmonella enterica serovar Typhi antibody titers between biliary tract cancer cases, biliary stone cases without evidence of cancer, and healthy subjects in a large population-based case-control study in Shanghai, China

A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella ; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE . Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA . Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75531/1/j.1365-2958.2002.02884.x.pd

Mouse antibody of IgM class is prone to non-enzymatic cleavage between CH1 and CH2 domains

Abstract IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo

Antimicrobial Resistance Incidence and Risk Factors among Helicobacter pylori–Infected Persons, United States

Helicobacter pylori is the primary cause of peptic ulcer disease and an etiologic agent in the development of gastric cancer. H. pylori infection is curable with regimens of multiple antimicrobial agents, and antimicrobial resistance is a leading cause of treatment failure. The Helicobacter pylori Antimicrobial Resistance Monitoring Program (HARP) is a prospective, multicenter U.S. network that tracks national prevalence rates of H. pylori antimicrobial resistance. Of 347 clinical H. pylori isolates collected from December 1998 through 2002, 101 (29.1%) were resistant to one antimicrobial agent, and 17 (4.8%) were resistant to two or more antimicrobial agents. Eighty-seven (25.1%) isolates were resistant to metronidazole, 45 (12.9%) to clarithromycin, and 3 (0.9%) to amoxicillin. On multivariate analysis, black race was the only significant risk factor (p < 0.01, hazard ratio 2.04) for infection with a resistant H. pylori strain. Formulating pretreatment screening strategies or providing alternative therapeutic regimens for high-risk populations may be important for future clinical practice

Emergence of new PCR ribotypes from the hypervirulent Clostridium difficile 027 lineage.

Clostridium difficile is the most common cause of antibiotic-associated diarrhoea worldwide. Over the past 10 years, the incidence and severity of disease have increased in North America and Europe due to the emergence of a hypervirulent clone designated PCR ribotype 027. In this study, we sought to identify phenotypic differences among a collection of 26 presumed PCR ribotype 027 strains from the US and the UK isolated between 1988 and 2008 and also re-evaluated the PCR ribotype. We demonstrated that some of the strains typed as BI by restriction endonuclease analysis, and presumed to be PCR ribotype 027, were in fact other PCR ribotypes such as 176, 198 and 244 due to slight variation in banding pattern compared to the 027 strains. The reassigned 176, 198 and 244 ribotype strains were isolated in the US between 2001 and 2004 and appeared to have evolved recently from the 027 lineage. In addition, the UK strains were more motile and more resistant to most of the antibiotics compared to the US counterparts. We conclude that there should be a heightened awareness of newly identified PCR ribotypes such as 176, 198 and 244, and that they may be as problematic as the notorious 027 strains