Can a humid storage environment of surgical instruments before reprocessing increase patient safety and durability of instruments?

BACKGROUND: National and international guidelines recommend reprocessing of medical instruments to commence as soon as possible post-surgery; furthermore, they recommend that transport and storage of surgical instruments postoperatively occurs in a moist, humid atmosphere. The concern is that a dry storage environment results in deterioration of instruments. AIM: To evaluate whether residual protein or corrosion is associated with storage environment (dry or humid), holding time or number of treatment cycles. METHODS: The range of protein residue and corrosion were tested on surgical instruments contaminated with human blood amended Enterococcus faecalis ATCC 29212. Subsequently instruments were stored for 6, 12 and 24 h in dry or humid conditions. After one, 25 and 50 reprocessing cycles, instruments were examined for protein residues using the o-phthaldialdehyde (OPA) method or corrosion using stereomicroscopy, scanning electron microscopy and energy dispersive spectroscopy. FINDINGS: Protein residue found on instruments was 21.5-54.0 µg and corrosion corresponded to 0-5% of the inspected area. No associations between storage environment and protein residue (adjusted mean difference = 0.48, 95% confidence interval: -0.42, 1.37, P=0.30) or corrosion (P=0.20) were identified. Higher numbers of treatment cycles showed higher amounts of corrosion (mean: 1cycle = 0.06%, 25cycles = 0.52% and 50cycles = 1.45%). In contrast, higher numbers of treatment cycles showed lower amounts of protein residue (P<0.001). We found both lower protein residue concentration and lower corrosion rating at 12 h compared with 6 and 24 h holding time. CONCLUSION: Cleanliness and durability of instruments before reprocessing seems not to be affected by storage environment or holding time but instead by number of treatment cycles.

Whole-genome sequencing of Campylobacter jejuni isolated from Danish routine human stool samples reveals surprising degree of clustering.

OBJECTIVES: Outbreaks of Campylobacter are traditionally considered to be rare; however, rather than being the true nature of the disease, this may reflect our present inability to detect them. The aim of this study was to determine the genetic and epidemiological degree of clustering among Campylobacter jejuni isolates from Danish patients. METHODS: Whole-genome sequencing (WGS) was applied to 245 C. jejuni isolates from patients with domestically acquired infection over a 9-month period in 2015 and 2016. RESULTS: WGS demonstrated that 62 of the 245 isolates (25%) clustered genetically. In total, 21 genetic clusters were identified of which four (18%) consisted of five isolates or more. Seventeen (81%) of the 21 genetic clusters were clustered in space and/or time. Of the 245 isolates, 49 (20%) were part of a temporal and/or geographical cluster. The identified clusters included two outbreaks; one which had not been identified through the existing surveillance system. CONCLUSIONS: Using WGS, we show that Campylobacter case clustering and even outbreaks appear to occur more often than previously assumed, providing important new insight into the relatively poorly understood epidemiology of the most important cause of bacterial gastroenteritis in the industrialized world.

First report of Actinomyces europaeus bacteraemia result from a breast abscess in a 53-year-old man.

This is the first report of Actinomyces europaeus bacteraemia in a 53-year-old man. The bacteraemia was the result of a breast abscess. Identification was established by matrix-assisted desorption ionization-time of flight mass spectrometry and confirmed by 16S rRNA gene sequencing. The patient was treated with surgical drainage and penicillin for 4 weeks; the patient did not experience any relapse during 6 months of follow-up.

High incidence of Campylobacter concisus in gastroenteritis in North Jutland, Denmark: a population-based study.

The incidence of non-thermophilic Campylobacter species was assessed in an unselected population-based study in a mixed urban and rural community in North Jutland, Denmark. In a 2-year study period, 11,314 faecal samples from 8302 patients with gastroenteritis were cultured with supplement of the filter method. We recovered a high incidence of Campylobacter concisus (annual incidence 35/100,000 inhabitants), almost as high as the common Campylobacter jejuni/coli. In contrast, there was a very low incidence of other non-thermophilic Campylobacter species, such as Campylobacter upsaliensis. Campylobacter concisus was, unlike C. jejuni/coli, found more frequently among small children (<1 year) and the elderly (≥ 65 years). Around 10% of the patients with C. consisus had co-infections dominated by Clostridium difficile and Salmonella enterica, whereas co-infections occurred in about 5% of C. jejuni/coli patients. We observed a seasonal variation in C. jejuni/coli with a peak incidence in late summer months and autumn, whereas there was an almost constant monthly prevalence of C. concisus. Among patients participating in a questionnaire sub-study, there was a higher degree of close contacts with animals, especially dogs, as well as a higher travel exposure among C. jejuni/coli patients compared with C. concisus patients. We did not culture any C. concisus in stool samples from a small cohort of healthy individuals. Future studies have to focus on the clinical follow-up and the long-term risk of inflammatory bowel diseases in C. concisus-positive patients. We conclude that there is a high incidence of C. concisus in Denmark.

Short-term and medium-term clinical outcomes of Campylobacter concisus infection.

There are only sparse data on the short-term and medium-term clinical impacts of Campylobacter concisus infection. A clinical study was performed during a 2-year period to determine the clinical manifestations in C. concisus-positive adult patients. A case patient was defined as an adult patient (≥18 years) with a C. concisus-positive stool sample during the study period. Clinical data were obtained with use of a questionnaire supplemented with the patients' medical records, if any. The short-term and medium-term clinical manifestations in these patients were compared with those of patients with Campylobacter jejuni/Campylobacter coli infection. One hundred and seventy-four C. concisus patients and 196 C. jejuni/C. coli patients participated in the study. Patients with pre-existing inflammatory bowel disease or microscopic colitis or enteric co-infection were excluded from review of the clinical manifestations. Comparison of the short-term clinical manifestations in 139 C. concisus patients with those in 187 C. jejuni/C. coli patients showed a significantly lower prevalence of fever, chills, mucus and blood in stools, and weight loss. However, 80% of C. concisus patients, but only 32% of C. jejuni/C. coli patients, had diarrhoea for >2 weeks. After a 6-month follow-up period, 12% of C. concisus patients were diagnosed with microscopic colitis, whereas no C. jejuni/C. coli patients were diagnosed with non-infective colitis. Irritable bowel symptoms were common in both groups at follow-up. C. concisus infection seems to cause a milder course of acute gastroenteritis than C. jejuni/C. coli infection, but is associated with more prolonged diarrhoea.

Drug resistance mutations and outcome of second-line treatment in patients with first-line protease inhibitor failure on nelfinavir-containing HAART.

OBJECTIVES: To determine resistance mutations emerging in HIV-1-infected patients experiencing their first protease inhibitor (PI)-failure on nelfinavir-containing highly active antiretroviral therapy (HAART), and to assess virological response to rescue regimens. METHODS: Plasma HIV-1 RNA from 24 patients failing nelfinavir-containing HAART was sequenced. Failure was defined as two consecutive measurements of viral load > 400 HIV-1 RNA copies/mL. Patients with previous failure on other PIs were excluded. Data on response to second-line treatment was extracted from patient files. RESULTS: At failure primary protease mutations were found in 14 patients (58%). Ten patients had D30N (38%), five patients had L90M (19%), two patients had V82A/F (8%) and two patients had M46I/L (8%). Two patients had both D30N and L90M. Pronounced increases of secondary protease mutations were seen at codon 88 (Delta: 33%), codon 36 (Delta: 30%) and codon 71 (Delta: 17%). Of eight patients with N88D, seven also harboured D30N (P < 0.01). Polymorphisms at codon 63 were detected at baseline in all patients who developed primary resistance mutations at failure (P < 0.01). On rescue regimens, 78% achieved viral loads below limit of detection (BLD). The presence of primary protease mutations was not associated with a higher risk of failure on second-line treatment. CONCLUSION: In patients failing nelfinavir-containing HAART, D30N was detected frequently and L90M occasionally. A pronounced accumulation of the secondary protease mutations N88D, M36I, and A71V/T was found, and D30N was strongly associated with N88D. A high proportion of patients became undetectable on second-line treatment and the presence of primary resistance mutations did not negatively affect the outcome of rescue regimens.

The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates.

A cDNA encoding CYP79B1 has been isolated from Sinapis alba. CYP79B1 from S. alba shows 54% sequence identity and 73% similarity to sorghum CYP79A1 and 95% sequence identity to the Arabidopsis T42902, assigned CYP79B2. The high identity and similarity to sorghum CYP79A1, which catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, suggests that CYP79B1 similarly catalyses the conversion of amino acid(s) to aldoxime(s) in the biosynthesis of glucosinolates. Within the highly conserved 'PERF' and the heme-binding region of A-type cytochromes, the CYP79 family has unique substitutions that define the family-specific consensus sequences of FXP(E/D)RH and SFSTG(K/R)RGC(A/I)A, respectively. Sequence analysis of PCR products generated with CYP79B subfamily-specific primers identified CYP79B homologues in Tropaeolum majus, Carica papaya, Arabidopsis, Brassica napus and S. alba. The five glucosinolate-producing plants identified a CYP79B amino acid consensus sequence KPERHLNECSEVTLTENDLRFISFSTGKRGC. The unique substitutions in the 'PERF' and the heme-binding domain and the high sequence identity and similarity of CYP79B1, CYP79B2 and CYP79A1, together with the isolation of CYP79B homologues in the distantly related Tropaeolaceae, Caricaceae and Brassicaceae within the Capparales order, show that the initial part of the biosynthetic pathway of glucosinolates and cyanogenic glucosides is catalysed by evolutionarily conserved cytochromes P450. This confirms that the appearance of glucosinolates in Capparales is based on a cyanogen 'predisposition'. Identification of CYP79 homologues in glucosinolate-producing plants provides an important tool for tissue-specific regulation of the level of glucosinolates to improve nutritional value and pest resistance.

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin.

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained. Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction. CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.

Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli.

The multifunctional tyrosine N-hydroxylase, cytochrome P450TYR (CYP79), from Sorghum bicolor catalyzing the conversion of tyrosine to p-hydroxyphenyl-acetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, has been expressed in Escherichia coli using the isopropyl-beta-D-thiogalactopyranoside-inducible vector pSP19g10L, containing the cDNA encoding CYP79. The expression construct was optimized by reducing the length of the N-terminal hydrophobic core of the signal sequence of cytochrome P450TYR and by exchanging the first eight codons with the first eight codons of bovine P45017 alpha. The highest yielding construct provided 200-500 nmol P450TYR/liter cell culture. The recombinant P450TYR was gently and efficiently extracted from E. coli spheroblasts by temperature-induced phase partitioning of Triton X-114 in the presence of 30% glycerol and isolated by DEAE and reactive red chromatography. In reconstitution experiments using saturating amounts of sorghum NADPH-cytochrome P450 reductase, the Km and turnover rate for isolated recombinant P450TYR was 0.22 +/- 0.06 mM and 49.2 +/- 3.8 min-1, respectively, whereas a turnover rate as high as 350 min-1, was obtained using E. coli membranes. Addition of 3 mM glutathione stimulated the activity of reconstituted P450TYR and of sorghum microsomes although the effect was highly variable. Phenylalanine, the precursor of several cyanogenic glucosides, gave a type I binding spectrum, but was not metabolized by P450TYR, demonstrating the high substrate specificity of this P450. Administration of radioactively labeled p-hydroxyphenylacetaldoxime to E. coli cells, showed E. coli metabolized p-hydroxyphenylacetaldoxime independent of the expression of P450TYR.

Pleuritis-pericarditis--an unusual initial manifestation of mixed connective tissue disease.

Cardiac involvement has been reported in mixed connective tissue disease (MCTD). We describe a 16-year-old girl in whom pleuritis and pericarditis occurred as the initial clinical manifestations of MCTD. Although pleuritis and pericarditis form a common clinical entity in MCTD, it is rarely seen as an initial manifestation. If MCTD is suspected, the diagnosis can be made by the clinical findings and the occurrence of a high titre of antibody against ribonuclease-sensitive ribonucleoprotein (RNP). This report emphasizes the importance of screening for connective tissue disease in patients with pericarditis/pleuritis.

[Klinefelter's syndrome associated with primary mediastinal germinal cell tumor]. / Klinefelter's syndrom associeret med primaer malign mediastinal germinalcelletumor.

A case of a man aged 25 year with Klinefelter's syndrome (KS) associated with a primary malignant germinal cell tumour is presented. Echocardiography was of assistance in the diagnosis as a tumour behind the heart was demonstrated. If patients with KS develop cardiopulmonary symptoms, referral for x-ray of the thorax is recommended. If there is enlargement of the heart or increase in width of the mediastinum, echocardiography should be performed. This case history emphasizes further that a negative cytological test does not disprove malignancy.

[Fatal thrombocytopenia triggered by quinidine]. / Kinidinudløst trombocytopeni med letalt forløb.

A woman aged 60 years receiving anticoagulation treatment on account of artificial mitral and aortic valve prostheses developed severe thrombocytopenia three weeks after the commencement of quinidine treatment. The results of investigations suggested severe thrombocytopenia precipitated by quinidine. The severe thrombocytopenia persisted despite intensive treatment and the condition ended fatally on account of cerebral haemorrhage. The combination of quinidine and oral anticoagulation treatment is not unusual and it is important to bear in mind that both of these preparations may cause increased haemorrhagic tendencies.

[Cauda equina syndrome developing during anticoagulant therapy]. / Cauda equina syndrom under antikoagulationsbehandling.

A woman aged 70 years with rheumatic heart disease developed the cauda equina syndrome (CES) seven days after commencement of anticoagulation therapy. Investigation suggested an intraspinal haematoma with subarachnoid haemorrhage. The patient was considered to be inoperable. The untreated CES resulted in permanent neurological deficits.

Mercury-selenium interactions in relation to histochemical staining of mercury in the rat liver.

Selenium has been suggested to enhance the histochemical staining of mercury when sections of tissue are subjected to the silver-enhancement method. In the present study, histochemical staining patterns of mercury in tissue sections of rat livers were compared with the actual content of organic and inorganic Hg in the livers, in both the presence and the absence of Se. Rats were injected intravenously with 5 micrograms of Hg g-1 body weight as methyl [203Hg] mercury chloride (MeHg) or as [203Hg]mercuric chloride (Hg2+). After 2 h, half the rats received an additional intraperitoneal injection of 2 micrograms of Se g-1 body weight as sodium [75Se]selenite. All the rats were killed 1 h later. Homogenized liver samples were prepared for mercury analysis by two different methods: alkaline digestion and ultrasonic disintegration. Quantitative chemical analysis based on benzene extraction of the radioactively labelled Hg compounds showed that the chemical form of mercury, either organic or inorganic, was preserved from its administration to its deposition in the liver. Light and electron microscopy demonstrated that no silver enhancement of Hg occurred when MeHg alone was present in the sections of tissue, whereas MeHg accompanied by Se induced a moderate deposition of silver grains. In contrast, sections containing Hg2+ alone yielded some staining, and the addition of Se increased the staining dramatically. The results of the present study show that acute selenite pretreatment is a prerequisite for the histochemical demonstration of methyl mercury, and greatly increases the staining of inorganic mercury when applying the silver-enhancement method.