An audit of Cryptosporidium and Giardia detection in Scottish National Health Service Diagnostic Microbiology Laboratories.

Giardia duodenalis and Cryptosporidium species are protozoan parasites capable of causing gastrointestinal disease in humans and animals through the ingestion of infective faeces. Whereas Cryptosporidium species can be acquired locally or through foreign travel, there is the mis-conception that giardiasis is considered to be largely travel-associated, which results in differences in laboratory testing algorithms. In order to determine the level of variation in testing criteria and detection methods between diagnostic laboratories for both pathogens across Scotland, an audit was performed. Twenty Scottish diagnostic microbiology laboratories were invited to participate with questions on sample acceptance criteria, testing methods, testing rates and future plans for pathogen detection. Reponses were received from 19 of the 20 laboratories representing each of the 14 territorial Health Boards. Detection methods varied between laboratories with the majority performing microscopy, one using a lateral flow immunochromatographic antigen assay, another using a manually washed plate-based enzyme immunoassay (EIA) and one laboratory trialling a plate-based EIA automated with an EIA plate washer. Whereas all laboratories except one screened every stool for Cryptosporidium species, an important finding was that significant variation in the testing algorithm for detecting Giardia was noted with only four laboratories testing all diagnostic stools. The most common criteria were 'travel history' (11 laboratories) and/or 'when requested' (14 laboratories). Despite only a small proportion of stools being examined in 15 laboratories for Giardia (2%-18% of the total number of stools submitted), of interest is the finding that a higher positivity rate was observed for Giardia than Cryptosporidium in 10 of these 15 laboratories. These findings highlight that the underreporting of Giardia in Scotland is likely based on current selection and testing algorithms.

Disparities in disease presentation in the four screenable cancers according to health insurance status.

OBJECTIVES: Current guidelines support the use of screening for early detection in breast, prostate, colorectal and cervical cancer. The purpose of this study was to evaluate whether insurance status predicts for more advanced disease in these four currently screened cancers. STUDY DESIGN: The Surveillance, Epidemiology, and End Results (SEER) database was queried for breast, prostate, colorectal and cervix in patients aged 18-64 years. The database was queried from 2007 to 2011, with 425,614 patients with known insurance status included. METHODS: Multinomial logistic regression was used to evaluate insurance status and cancer presentation. RESULTS: Under multivariate analysis for breast cancer, uninsured patients more often had invasive disease (odds ratio [OR]: 1.55), T- (OR: 2.00), N- (OR: 1.59) stage, and metastatic disease (OR: 3.48), and were more often high-grade (OR: 1.21). For prostate cancer, uninsured patients again presented more commonly with higher T-stage (OR: 1.45), nodal (OR: 2.90) and metastatic (OR: 4.98) disease, in addition to higher prostate-specific antigen (OR: 2.85) and Gleason score (OR: 1.65). Colorectal cancer had similar findings with uninsured individuals presenting with more invasive disease (OR: 1.78), higher T (OR: 1.86), N (OR: 1.22), and M (OR: 1.58) stage, in addition to higher carcinoembryonic antigen levels (OR: 1.66). Similar results were seen for cervical cancer with uninsured having higher T (OR: 2.03), N (OR: 1.21), and M (OR: 1.45) stage. CONCLUSION: In the four cancers detected by screening exams, those without health insurance present with more advanced disease, with higher stage and grade, and more elevated tumour markers.

The impact of postmastectomy and regional nodal radiation after neoadjuvant chemotherapy for clinically lymph node-positive breast cancer: a National Cancer Database (NCDB) analysis.

BACKGROUND: Following neoadjuvant chemotherapy (NAC), the optimal strategies for postmastectomy radiotherapy (PMRT) and regional nodal irradiation (RNI) after breast-conserving surgery (BCS) are controversial. In this analysis, we evaluate the impact of these radiotherapy (RT) approaches for women with clinically node-positive breast cancer treated with NAC in the National Cancer Database (NCDB). PATIENTS AND METHODS: Women with cT1-3 cN1 M0 breast cancer treated with NAC were divided into four cohorts by surgery [Mastectomy (Mast) versus BCS] and post-chemotherapy pathologic nodal status (ypN0 versus ypN+). Overall survival (OS) was estimated using the Kaplan-Meier method and RT approaches were analyzed using the log-rank test, multivariate Cox models, and propensity score-matched analyses. RESULTS: From 2003 to 2011, 15 315 cases were identified including 3040 Mast-ypN0, 7243 Mast-ypN+, 2070 BCS-ypN0, and 2962 BCS-ypN+ patients. On univariate analysis, PMRT was associated with improved OS for both Mast-ypN0 (P = 0.019) and Mast-ypN+ (P < 0.001) patients. On multivariate analyses adjusted for factors including age, comorbidity score, cT stage, in-breast pathologic complete response, axillary surgery, ypN stage, estrogen receptor status and hormone therapy, PMRT remained independently associated with improved OS among Mast-ypN0 [hazard ratio (HR) = 0.729, 95% confidence interval (CI) 0.566-0.939, P = 0.015] and Mast-ypN+ patients (HR = 0.772, 95% CI 0.689-0.866, P < 0.001). No differences in OS were observed with the addition of RNI to breast RT for BCS-ypN0 or BCS-ypN+ patients. Propensity score-matched analyses demonstrated identical patterns of significance. On subset analysis, OS was improved with PMRT in each pathologic nodal subgroup (ypN0, ypN1, and ypN2-3) (all P < 0.05). CONCLUSIONS: In the largest reported analysis of RT for cN1 patients treated with NAC, PMRT was associated with improved OS for all pathologic nodal subgroups. No OS differences were observed with the addition of RNI to breast RT.

A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-oncology patients.

A blinded prospective study was performed to determine whether screening of whole blood using a real-time, panfungal polymerase chain reaction (PCR) technique could predict the development of invasive fungal infection (IFI) in immunocompromised haemato-oncology patients. In all, 78 patients (125 treatment episodes) were screened twice weekly by real-time panfungal PCR using LightCyclertrade mark technology. IFI was documented in 19 treatment episodes (five proven, three probable and 11 possible), and in 12, PCR was sequentially positive. PCR positivity occurred in: 4/5 proven; 2/3 probable; 6/11 possible; and 29/106 with no IFI. In 8/12 with IFI and sequentially positive PCR results, PCR positivity occurred before (median 19.5 days) and in 4/12 (median 10.5 days) after the initiation of empirical antifungal therapy. Based on sequential positive results for proven/probable IFI sensitivity, specificity, positive predictive value and negative predictive value were 75, 70, 15 and 98%, respectively. Real-time panfungal PCR is a sensitive tool for the early diagnosis of IFI in immunocompromised haemato-oncology patients. It may be most useful as a screening method in high-risk patients, either to direct early pre-emptive antifungal therapy or to determine when empirical antifungal therapy can be withheld in patients with antibiotic--resistant neutropenic fever. However, these strategies require further assessment in comparative clinical trials.

Purification and partial characterization of a second cysteine proteinase inhibitor from ungerminated barley (Hordeum vulgare L.).

It was previously shown that ungerminated barley contains inhibitors that suppress the activities of green malt cysteine proteinases. This paper reports the purification and partial characterization of a second barley cysteine endoproteinase inhibitor, a protein called lipid transfer protein 2 (LTP2). The chromatographically purified inhibitor had a molecular mass of 7112. The amino acid composition and sequence data of the purified inhibitor indicated that it was a protein whose gene, but not the protein itself, was isolated earlier from barley aleurone tissue. The purified protein inhibited the activities of electrophoretically separated green malt cysteine proteinases but not the activities of the serine- or metalloproteinases. The purified LTP2 inhibited the same proteases as the LTP1 that was characterized previously but was present in the mature seed in much smaller amounts. Neither LTP1 nor LTP2 has been proven to transport lipids in vivo, and it seems possible that both serve to keep cysteine endoproteinases that are synthesized during barley seed development inactive until the plant needs them. The small amount of LTP2 in the seed made it impossible to determine whether it, like LTP1, is involved in beer foam formation. Because of its proteinase-inhibiting ability and its resistance to heat inactivation, some of the LTP2 may persist in beer.

Control of an outbreak of respiratory syncytial virus infection in immunocompromised adults.

Respiratory syncytial virus (RSV) is increasingly recognized as an important pathogen in immunocompromised adults, particularly those receiving bone marrow transplants, and, given the ease with which it spreads, represents a significant nosocomial problem. We describe an outbreak of RSV infection involving eight patients on a haematology/oncology ward which was controlled by early screening of patients and staff. Positive patients were cohort nursed on a separate ward and basic infection control measures including use of gowns and gloves were enforced. Children under age 12 were denied ward access. All patients with lower respiratory tract infection, and bone marrow transplant recipients with upper respiratory symptoms, were treated with nebulized ribavirin. There were no deaths. We conclude that awareness of the risk of RSV infection in immunocompromised patients coupled with rapid diagnosis and treatment, screening of symptomatic patients and staff, cohort nursing of cases and basic infection control procedures can prevent spread of RSV infection and reduce morbidity.

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley.

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S2 site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1-9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.

Appearance of Endoproteolytic Enzymes during the Germination of Barley.

Barley endoproteolytic enzymes are important to germination because they hydrolyze endosperm storage proteins to provide precursors for new protein synthesis. We recently developed an electrophoretic method utilizing gel-incorporated protein substrates to study the endoproteinases of 4-d-germinated barley (Hordeum vulgare L. cv Morex) grain. This work extends those findings to determine the temporal pattern of the appearance of the endoproteinases during germination, the sensitivities of the proteinases to class-specific proteinase inhibitors, and where, in germinating caryopses, the proteinases reside. Six endoproteinase activity bands (representing a minimum of seven enzymes) were present in 5-d-germinated barley grain extracts subjected to electrophoresis in nondenaturing gels at pH 8.8. The activities of two of the enzyme bands ("neutral" proteinases) increased as the pH was increased from 3.8 to 6.5. The activities of the remaining four ("acidic") bands diminished abruptly as the pH increased above 4.7. Two proteinase bands hydrolyzed gelatin but not edestin, four of the proteinases hydrolyzed both gelatin and edestin at nearly the same rates, and one enzyme degraded only edestin. One neutral endoproteinase was sensitive to diisopropyl fluorophosphate inhibition, and the other was not inhibited by any of inhibitors tested. Four of acidic enzymes were cysteine proteinases [inhibited by trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and N-ethylmaleimide]; the other was an aspartic acid endoproteinase (sensitive to pepstatin). Only the aspartic proteinase was detected in either ungerminated or steeped barley grain. During the germination (malting) process, the aspartic endoproteinase activity decreased until the second day of germination and then increased until germination day 5. The first endoproteinase(s) induced during germination was a neutral enzyme that showed activity on the 1st day of the germination phase after steeping. Most of the endoproteinases became active on the 2nd or 3rd germination day, but one cysteine proteinase was not detected until the 5th day. Acid cysteine proteinases were present in the aleurone, scutellum, and endosperm tissues but not in shoots and roots. The aleurone layer and endosperm contained almost exclusively band B1 neutral proteinases, whereas the scutellum, shoots, and roots contained both B1 and B2 bands. This work shows that germinating barley contains a complex set of proteinases whose expression is temporally and spatially controlled. But, at the same time, it also shows that this electrophoretic method for separating and studying individual enzymes of this complex will allow us to more readily characterize and purify them.

A survey of oral and maxillofacial surgeons concerning their knowledge, beliefs, attitudes, and behavior relative to parameters of care.

In December 1990, a survey was sent to 1,296 randomly selected members of the American Association of Oral and Maxillofacial Surgeons (AAOMS) to determine their previous experience with standards and criteria of care, their type of practice, the educational methods that influence their professional decisions, and their attitudes about the development and use of parameters of care. A 55.7% response was obtained. This article reports the results of this survey. The average age of responding surgeons was 45 years and they had been in practice an average of 15 years. The majority were in private practice, had hospital staff privileges, worked between 31 and 55 hours per week, spent 90% of their working week in direct patient care, and devoted an average of 9.16 hours per month to professional affairs outside of their practice. Forty-two percent (42%) of the practitioners were in solo practice, whereas 50% practiced in groups. Surgeons concentrated 65% of their patient care time on dentoalveolar surgery and a significant number planned increases in practice activity in implant, orthognathic, and temporomandibular joint surgery. They learned new clinical skills in various ways, there being a difference between the most convenient and effective methods of learning. A majority of surgeons had been involved with quality assurance activities in the past 5 years. They were predominantly favorable to parameters now and when they first learned about them, but few thought they had a clear understanding about how parameters of care would be used.(ABSTRACT TRUNCATED AT 250 WORDS)

A Proteinase from Germinated Barley : II. Hydrolytic Specificity of a 30 Kilodalton Cysteine Proteinase From Green Malt.

The hydrolytic specificity of a 30 kilodalton cysteine proteinase purified from germinated barley (Hordeum vulgare L. cv Morex) was investigated using high performance liquid chromatography to characterize its hydrolysis of two small barley seed proteins, the alpha- and beta-hordothionins. The reduced and pyridylethylated thionins were rapidly cleaved, resulting in the production of a limited number of peptides. Peptide bonds Gly9-Arg10, Cys 16-Arg17, Cys25-Ala26, and Thr34-Ser35 were most susceptible to hydrolysis, the peptide bonds Arg5-Ser6, Arg19-Gly20 in both thionins and Lys38-Cys39 in beta-hordothionin and Cys29-Arg30 of alpha-hordothionin being broken at much slower rates. The hydrolysis patterns were highly reproducible from assay to assay and with various enzyme preparations. The specificity was apparently defined by the amino acids in the P(2) position, not those immediately adjacent to the susceptible bonds. The P(2) amino acid residues of the released peptides were always either leucine, valine, tyrosine, or pyridylethylcysteine. From these observations and from the rates of release of the various peptides, it appears that the barley 30 kilodalton endoproteinase has an S2 subsite that preferentially binds the leucine side chain: i.e. for hydrolyzing the peptide bond P(1)-P(1)' in the general sequence NH(2)-P(2)-P(1)-P(1)'-COOH, the enzyme is selective for leucine and, to a lesser extent, valine and tyrosine at position P(2). The barley proteinase thus resembles two other cysteine proteinases, papain and Streptococcal proteinase, in its specificity.

A Proteinase from Germinating Barley : I. Purification and Some Physical Properties of a 30 kD Cysteine Endoproteinase from Green Malt.

A proteinase was purified from germinated barley (green malt from Hordeum vulgare L. cv Morex) by acidic extraction, ammonium sulfate fractionation and successive chromatographies on CM-cellulose, hemoglobin sepharose, Sephadex G-75 and organomercurial agarose columns. The overall purification and final recovery were 290-fold and 7.5%, respectively. The purified enzyme was homogeneous on analytical gel electrophoresis, yielding a single protein associated with protease activity. An apparent molecular weight of about 20 kilodaltons was estimated for the native enzyme from gel filtration. SDS-gel electrophoresis revealed a single polypeptide of about 30 kilodaltons. The optimum pH for the hydrolysis of hemoglobin was around 3.8. The enzyme was strongly inhibited by leupeptin but was insensitive to phenylmethylsulfonyl fluoride, indicating that it was a cysteine proteinase. It hydrolyzed several large proteins from various origins. The ability of the enzyme to digest barley storage proteins in vitro was examined using SDS-gel electrophoresis. The hydrolysis patterns obtained showed that the enzyme rapidly hydrolyzed the large hordein polypeptides into relatively small fragments. The results of this study suggest that this 30 kilodalton enzyme is one of the predominant cysteine proteinases secreted into the starchy endosperm during barley germination and that it plays a major role in the mobilization of storage proteins.

Neurodevelopmental and respiratory outcome in early childhood after human surfactant treatment.

We assessed postnatal growth, neurodevelopmental outcome, and occurrence of respiratory illnesses in 46 infants of very low birth weight who were enrolled in a randomized, controlled, bicenter clinical trial of human surfactant treatment for respiratory distress syndrome. No long-term adverse effects of human surfactant treatment were detected between control and human surfactant-treated infants with respect to growth, neurologic, or developmental outcome. Infants with chronic lung disease, regardless of treatment group, had poorer growth and were more likely to have neurodevelopmental abnormalities at 12 to 24 months of age.

Biochemical data bearing on the relationship between the genome of Triticum urartu and the A and B genomes of the polyploid wheats.

To determine whether the Triticum urartu genome is more closely related to the A or B genome of the polyploid wheats, the amino acid sequence of its purothionin was compared to the amino acid sequences of the purothionins in Triticum monococcum, Triticum turgidum, and Triticum aestivum. The residue sequence of the purothionin from T. urartu differs by five and six amino acid substitutions respectively from the alpha 1 and alpha 2 forms coded for by genes in the B and D genomes, and is identical to the beta form specified by a gene in the A genome. Therefore, the T. urartu purothionin is either coded by a gene in the A genome or a chromosome set highly homologous to it. The results demonstrate that at least a portion of the T. urartu and T. monococcum genomes is homologous and probably identical. A variety of other studies have also shown that T. urartu is very closely related to T. monococcum and, in all likelihood, also possesses the A genome. Therefore, it could be argued that either T. urartu and T. monococcum are the same species or that T. urartu rather than T. monococcum is the source of the A genome in T. turgidum and T. aestivum. Except for Johnson's results, our data and that of others suggest a revised origin of polyploid wheats. Specifically, the list of six putative B genome donor species is reduced to five, all members of the Sitopsis section of the genus Aegilops.

Inhibition of growth of Rhizobium japonicum by cyclic GMP.

Exogenous cyclic guanosine-3',5'-monophosphate (cGMP) inhibited the growth of Rhizobium japonicum at less than 100 microM. Other nucleotides, including cyclic AMP, cyclic IMP, and cyclic CMP, had no inhibitory effect even at higher concentrations nor was the inhibition by cGMP reversed by cyclic AMP. The inhibitory effect was independent of the carbon and nitrogen source(s) used. cGMP did not inhibit the growth of any other species of bacterium tested, including several fast-growing Rhizobium species. The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition. Subsequently, growth resumed at a slower rate. Resumption of growth was not due to destruction of the nucleotide. Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions. No effect of cGMP on the derepression of respiratory nitrate reductase was observed.

Proton magnetic resonance studies of barley and wheat thionins: structural homology with crambin.

The thionins comprise a group of very basic proteins of Mr approximately 5000 found in the seeds of Gramineae. They each contain 45 amino acid residues arranged along a single polypeptide chain that is constrained by four disulfide bridges. Five thionins of known sequence, from barley and wheat, have been investigated and compared by 1H NMR spectroscopy at 600 MHz. From their spectral characteristics it is concluded that the five proteins have very similar, nonrandom conformations in 2H2O solution. Moreover, on the basis of selective nuclear Overhauser experiments at 300 MHz, features of their secondary and tertiary structures are shown to be similar to those of crambin, a related, hydrophobic protein extracted from seeds of the crucifer Crambe abyssinica. The strong compositional homology of the thionins facilitates the assignment of methyl and aromatic resonances, as only a few residues are replaced and these are at known sites. The substitution of leucine for an isoleucine does not affect significantly the local magnetic environment, suggesting that those isomeric side chains easily accommodate the same spatial constraints. A fast hydrogen-deuterium exchange is observed at pH 6.25, 25 degrees C. This indicates that, although of folded conformation, the thionins are structurally flexible polypeptides that efficiently expose all amides to the solvent.

The amino acid sequence of wheat beta-purothionin.

The complete amino acid sequence of beta-purothionin, a low molecular weight, very basic, protein isolated from wheat endosperm material, has been determined. beta-purothionin is toxic to some bacteria, to yeasts, and to animals when injected. The protein contains 45 amino acid residues and has a molecular weight of 4913. The 8 cysteine and 10 basic residues are distributed throughout the molecule. The primary structure of the protein shows considerable homology to those of the viscotoxins, which are toxic, small, basic proteins found in the leaves and stems of European mistletoe (Viscum album L.).