Highly resistant gram-negative microorganisms: incidence density and occurrence of nosocomial transmission (TRIANGLe Study).

OBJECTIVES: The objectives of this study were to determine the incidence density and the occurrence of horizontal spread of highly resistant gram-negative rods (HR-GNRs) in Dutch hospitals. The factors that influence these outcome measures were also investigated. METHODS: All patients with HR-GNRs, as determined by sample testing, who were hospitalized in 1 of 18 hospitals during a 6-month period (April through October 2007) were included in this study. For all available isolates, the species was identified, susceptibility was determined (including the presence of extended-spectrum ß-lactamases [ESBLs]), and molecular typing was performed. On the basis of a combination of species identification, molecular typing, and epidemiological data, the occurrence of nosocomial transmission was determined. RESULTS: The mean incidence density of patients with HR-GNRs was 55 per 100,000 patient-days (cumulative incidence, 39 per 10,000 patients admitted). A facility being a university hospital was a statistically significant (P = .03) independent determinant of a higher incidence of patients with HR-GNRs. The majority of HR-GNR isolates were ESBL producers. The adjusted transmission index-the ratio between secondary and primary cases-in the participating hospitals ranged from 0.0 to 0.2. The overall adjusted transmission index of HR-GNRs was 0.07. No determinants for a higher transmission index were identified. DISCUSSION: The nosocomial transmission rate of HR-GNRs was relatively low in all hospitals where well-established transmission-based precautions were used. The incidence density of patients with HR-GNRs was higher in university hospitals, probably due to the patient population and the complexity of the care provided.

Characteristics and incidence of Clostridium difficile-associated disease in The Netherlands, 2005.

During a 2-month period in 2005, 13 laboratories participated in a surveillance study of Clostridium difficile-associated disease (CDAD) in 17 hospitals in The Netherlands. The median incidence rate of CDAD was 16/10 000 patient admissions (2.2/10 000 patient-days) and varied from 1 to 46/10 000 patient admissions according to hospital. In total, 81 patients with CDAD were reported; 49 (61%) patients had nosocomial CDAD, and 29 (36%) patients were admitted to hospital when already suffering from diarrhoea. Two (2%) deaths were attributable to CDAD; both of these patients were admitted with severe community-onset CDAD and were aged >80 years. Among 64 toxinogenic isolates, ten (16%) belonged to PCR ribotype 027 and ten (16%) to PCR ribotype 014. Type 027 was identified in ten patients from one hospital during an unrecognised outbreak. Toxinotyping of the 64 isolates revealed the presence of six different toxinogenic types, with 41 (64%) isolates of toxinotype 0, ten (16%) isolates of toxinotype III, and nine (14%) isolates of toxinotype V. Of the 64 toxinogenic isolates, seven (11%) had a 39-bp deletion in the tcdC gene, 11 (17%) had an 18-bp deletion, and one (1%) had a deletion of c. 44 bp. Genes for binary toxin were present in 21 (33%) of the 64 toxinogenic isolates, mainly associated with toxinotypes III and V. It was concluded that the median CDAD incidence rate of 16/10 000 patient admissions in The Netherlands is considerably lower than that in Canada and the USA, and that the emerging type 027 can spread unnoticed. The high proportion (36%) of CDAD cases with a community onset has important implications for future studies of the epidemiology of CDAD.

[Two hospital staff with throat carriage of methicillin-resistant Staphylococcus aureus, which had to be treated with systemic antibiotics]. / Twee ziekenhuismedewerkers met keeldragerschap van meticillineresistente Staphylococcus aureus waarvoor systemische antibiotische behandeling noodzakelijk was.

Two hospital staff, women aged 20 and 22 years, were inadvertently found to be positive for methicillin-resistant Staphylococcus aureus (MRSA). Both had been in a hospital outside of the Netherlands, but due to the long period of time that had elapsed since then, they did not fall under the standard protocol for MRSA screening. After the usual wash procedure with chlorhexidine and mupirocin nasal ointment treatment, they remained positive for MRSA in the throat culture. Both patients still had their pharyngeal tonsils and were suffering from throat complaints. After systemic treatment with two antibiotics, they both became MRSA-free. Throat carriership of MRSA might be a reason why MRSA eradication fails in the case of apparently healthy healthcare workers. The addition of a throat culture to the screening of healthcare workers would therefore be useful.

Source, carriers, and management of a Serratia marcescens outbreak on a pulmonary unit.

An outbreak of Serratia marcescens was seen on a pulmonary ward from September 1999 until September 2000. During this period, there were two distinct clusters of S. marcescens isolation. In the first episode, September-October 1999, S. marcescens isolates with the same resistance pattern were isolated in 10 patients. PFGE (pulsed-field gel electrophoresis) following digestion with SpeI confirmed that these isolates were identical. After an initial decline in the number of isolates, the incidence rose again in March 2000. The resistance pattern of these isolates differed from that in 1999. PFGE showed that most of the isolates in 2000 were identical and had replaced the previous strain (strain 1). In the second episode, January-August 2000, 26 patients were colonized with the subsequent strain (strain 2). Three of these patients had serious clinical problems due to S. marcescens, two had bacteraemia and one empyema. In September 2000, strain 2 was also detected in stock solutions for inhalation therapy. After discontinuation of the use of stock solutions and emphasizing hygienic measures, the outbreak resolved. The majority (68%) of the patients positive for S. marcescens suffered from COPD (chronic obstructive pulmonary disease). PFGE results suggest that several COPD patients were carriers of the same strain of S. marcescens for a prolonged time. Re-admission of these patients could have lead to re-introduction of the epidemic strains.

Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain.

A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital. Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea. Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1). Relapses occurred in three patients. Nineteen of the 27 episodes required anti-Clostridium difficile treatment. Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical. The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied. No phenotypic activity of enterotoxin A was detected. A deletion of approximately 1.7 kb was found in the toxin A gene. Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin.

Apparent culture-negative prosthetic valve endocarditis caused by Peptostreptococcus magnus.

In two patients with prosthetic valve endocarditis due to Peptostreptococcus magnus, blood cultures in the BacT/Alert and BACTEC 9240 systems were signal negative. The capability of the BacT/Alert system to detect various Peptostreptococcus species was assessed. P. magnus and P. anaerobius could not be detected, and subcultures remained negative. The growth in conventional media of these two species and other Peptostreptococcus species was similar.

An insulin receptor mutant (Asp707 --> Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin.

We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) alpha subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to alpha and beta subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant alpha subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR alpha and beta subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor alpha subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the alpha subunit carboxyl terminus to insulin binding.

Alternative splicing of the insulin receptor isoforms is altered in patients with leprechaunism.

Leprechaunism is a syndrome of severe insulin resistance. In general, mutations in both alleles of the insulin receptor are required for developing the leprechaun phenotype. Recently, we described a leprechaun patient having an Arg for Gly substitution in one allele of the insulin receptor whereas the other allele has the normal sequence. To explain the leprechaun phenotype, we searched for additional defects at the receptor level. The insulin receptor exists as two splice variants, either with (B form) or without exon 11 (A form). It has been suggested that a decrease in the relative amount of the A isoform can contribute to the development of an insulin resistant state. Fibroblasts from the leprechaun patient show levels of the A isoform of less than 20% whereas in control fibroblasts approximately 50% of the A isoform is present. However, in fibroblasts from two other patients with severe insulin resistance where the disease has been demonstrated to result from two mutated insulin receptor alleles, the same decline of the A isoform was seen. We conclude that the decrease in the relative amount of the A isoform in these patients is probably an epiphenomenon due to impaired insulin action.

A mutation in the insulin receptor that impairs proreceptor processing but not insulin binding.

Here we report the identification of a new mutation in the alpha-chain of the insulin receptor, changing Trp412 into Ser using DNA from consanguineous parents who gave birth to a child with leprechaunism. The mutant receptor was expressed stably in CHO and transiently in COS-1 cells. It was found that the Ser412 mutant is not cleaved into alpha- and beta-subunits and remains as a 210-kDa proreceptor at an intracellular site. This property of the mutant receptor is in line with the observed decreased insulin binding to the parental fibroblasts. Cross-linking experiments show that the Ser412 proreceptor is able to bind insulin with an affinity comparable to that of the wild-type alpha-chain. Despite its capacity to bind insulin, the mutant receptor is not autophosphorylated. We postulate that the patient was homozygous for the Trp412-->Ser mutation and that the mutation was responsible for the leprechaun phenotype. This is the first description of a transport-defective receptor with the mutation outside the tetrabasic processing site and a functional insulin binding domain. The ability of the Ser412 mutant to bind insulin in cross-linking experiments suggests that the impaired transport of the proreceptor to the cell surface is the primary cause for the binding defect to intact cells.

An in-frame insertion in exon 3 and a nonsense mutation in exon 2 of the insulin receptor gene associated with severe insulin resistance in a patient with Rabson-Mendenhall syndrome.

We have studied the structure and function of the insulin receptor in a patient (PK) with severe insulin resistance and Rabson-Mendenhall syndrome. Insulin binding to cultured fibroblasts from PK was almost not detectable and insulin-induced insulin receptor autophosphorylation and glucose uptake was abolished. The structure of the receptor gene was analysed by sequencing amplified products of the 22 exons with the flanking intron regions directly as well as after subcloning in pUCBM20 plasmids. Two mutant alleles of the insulin receptor gene were detected. One allele contains in-frame 12 additional base pairs in exon 3 coding for the amino acids Leu-His-Leu-Val located between Asp-261 and Leu-262 in the receptor's extracellular domain, being the first report of an insertion mutation of the insulin receptor gene. In the other allele Arg-86 in exon 2 is changed into a stop codon. Therefore, PK is compound heterozygous at the insulin receptor locus. Direct cDNA sequencing indicates that both mutant alleles are expressed in the patient's fibroblasts. Studies of the parents' fibroblasts revealed that PK inherited the insertion mutation from the father and the nonsense mutation from the mother. Insulin binding to fibroblasts of the mother was reduced (63% of control cells) and hormone binding to the father's cells shows a larger reduction (37% of control cells), but less severe than the patient's cells (11% of control). This investigation provides further evidence that the Rabson-Mendenhall syndrome is causally related to mutations in the insulin receptor gene.

Patients with lipodystrophic diabetes mellitus of the Seip-Berardinelli type, express normal insulin receptors.

Lipodystrophic diabetes mellitus of the Seip-Berardinelli type is a syndrome associated with insulin resistance and recessive inheritance. We have examined whether mutations in the insulin receptor are pathogenetic factors in this syndrome. Fibroblasts from three different patients with Seip-Berardinelli's lipodystrophy were tested for insulin binding, and insulin-stimulated receptor autophosphorylation. In addition, the coding region of both alleles of the iinsulin receptor gene was sequenced. No abnormalities in the number of high affinity insulin binding sites, and insulin-stimulated receptor autophosphorylation were detected. The insulin receptor related insulin-like growth factor I receptor also showed no functional changes. DNA sequence analysis of the amplified exons of the insulin receptor gene showed a silent mutation in patient 1 at codon Ser339, changing AGT to AGC. In patient 2 a heterozygous Met for Val substitution at position 985 was detected, which is a rare polymorphism. In patient 3 no mutations, other than described polymorphisms, were observed. These findings demonstrate that the primary genetic lesion in Seip-Berardinelli's lipodystrophy is outside the insulin receptor gene and that an involvement of the insulin-like growth factor I receptor is also unlikely.

An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport.

In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.

A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface.

We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface.

Methimazole increases thyroid-specific mRNA concentration in human thyroid cells and FRTL-5 cells.

Methimazole (1-methyl-2-mercaptoimidazole; MMI) increases thyroglobulin mRNA and thyroid peroxidase mRNA concentration in human thyroid cells and in FRTL-5 cells. MMI (1-10,000 microM) gives a dose-dependent increase of thyroglobulin concentration in the medium of human thyroid cells and FRTL-5 cells. The stimulation by MMI has no effect on the TSH-induced cAMP production and occurs in the presence or absence of thyrotropin (TSH). TSH increases the thyroglobulin and thyroid peroxidase mRNA synthesis in human thyroid cells and FRTL-5 cells. The accumulation of thyroglobulin in the medium has an optimum at 100 microU TSH/ml in FRTL-5 cells. This optimum can also be found in most human thyroid cell cultures.

Platelet volume analysis in thrombocytopenia in relation to bleeding tendency.

It has been shown that large platelets are hemostatically more active than the smaller ones. We therefore studied the relationship between the mean platelet volume and the percentage of micro- and mega-thrombocytes measured by a Coulter counter S plus II, and the bleeding tendency in 57 unselected patients with a platelet count below 50 X 10(9)/l. We found no significant differences for any of these parameters between patients without and those with mild or severe bleeding tendency. This also held true when patients with a possible platelet dysfunction or with coagulation abnormalities were excluded. We conclude that platelet volume analysis in unselected patients with severe thrombocytopenia is not helpful in the prediction of their risk of bleeding.