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Osteochondral lesions (OCL) are common among young patients and often require surgical interventions since cartilage has a poor capacity for self-repair. Bone marrow stimulation (BMS) has been used clinically for decades to treat OCLs, however a persisting challenge with BMS and other cartilage repair strategies is the inferior quality of the resulting fibrocartilaginous repair tissue. Lubrication-based therapies have the potential to improve the quality of cartilage repair tissue as joint lubrication is linked to local cartilage tissue strains and subsequent cellular responses including death and apoptosis. Recently, a full length recombinant human lubricin (rhLubricin) was developed and has been shown to lower friction in cartilage. This study investigated the effect of a single delayed injection of rhLubricin on cartilage repair in an in vivo rabbit OCL model using gross macroscopic evaluation, surface profilometry, histology, and tribology. Moderate improvement in macroscopic scores for cartilage repair were observed. Notably, quantitative analysis of Safranin-O histology showed that rhLubricin treated joints had significantly higher glycosaminoglycan content compared to saline treated joints, and there were no differences in repair integration between groups. Furthermore, rhLubricin treated joints had significantly lower friction coefficients tested across three sliding speeds compared to saline treated joints (rhLubricin: 0.15 ± 0.03 at 0.1 mm/s to 0.12 ± 0.03 at 10 mm/s, Saline: 0.22 ± 0.06 at 0.1 mm/s to 0.19 ± 0.05 at 10 mm/s). Overall, a single delayed injection of rhLubricin improved the quality and lubricating ability of the repair cartilage tissue without inhibiting repair tissue integration., Competing Interests: Conflicts of Interest Dr. Bonassar is a co-founder of and hold equity in 3DBio Corp. Dr. Colville, Dr. Reesink and Dr. Paszek are named inventors in pending patents related to the design and manufacturing of rhLubricin, assigned to Cornell University.

Background: Type 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum., Methods: An in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells., Results: The in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells., Conclusion: Rosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells., Competing Interests: Declaration of competing interest No conflicts., (Copyright © 2021 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

A systematic literature search was performed using the PubMed, MEDLINE, and the Cochrane Library databases according to the Preferred Reporting Items for Systematic review and Meta-Analyses guidelines on May 20, 2019. The keywords used were: ankle, distal tibia, distal fibula, fracture, arthroscopic, cartilage, and chondral. The objective of this study is to systematically review the characterization of intra-articular chondral injuries of the talus, tibial plafond, medial malleolus, and lateral malleolus in patients who undergo ankle arthroscopy following ankle fracture. Studies evaluating the incidence of chondral lesions at the time of arthroscopy for ankle fractures within any timeframe were included. The incidence of intra-articular chondral lesions was recorded, the location within the ankle, ankle fracture type, time of arthroscopy, characterization of chondral injury, complications, and outcome if available. Fifteen studies with 1355 ankle fractures were included. About 738 demonstrated evidence of chondral or osteochondral lesion (54.5%). Statistical analyses were carried out with statistical software package SPSS 24.0 (SPSS, Chicago, IL). We compared incidence rates of chondral injury based on Weber classification, malleolar fracture type, and Lauge-Hansen classification, using Pearson chi-square test. For all analyses, p < .05 was considered statistically significant. We found a high incidence of intra-articular chondral lesion in the setting of ankle fractures as demonstrated by arthroscopy., (Copyright © 2021 the American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Aspiration of the olecranon bursa is a treatment option for acute olecranon bursitis (OB). Typically, the aspirate is sent for microbiologic analysis, cell count, and crystal analysis. This study investigates the utility of fluid aspirate analysis from patients with clinically diagnosed aseptic OB., Methods: In this prospective study (IRB #i20-00986), patients presenting with acute aseptic OB were treated with aspiration as standard of care. Patients consented to participate in this study via phone. Patients with suspected infectious bursitis, open draining wound, and chronic OB were excluded. The aspirate was sent out for routine microbiologic analysis (aerobic and anaerobic cultures and Gram staining) and fluid analyses, including cell count with differential and crystal analysis. Nucleated and differential cell count was reported as absolute numbers per cubic millimeter and percentage, respectively. Compression wrap was applied after OB aspiration, and patients were asked to ice and take anti-inflammatory medications. Clinical follow-up was done after 6 weeks and at 3 months for resolution vs. recurrence of symptoms, and the mean time to resolution was reported., Results: A total of 26 patients (28 cases) with aseptic OB were enrolled in this study. Two patients had bilateral OB. The mean time to aspiration after the onset of symptoms was 26.4 days. One patient had recurrence of swelling after the first aspiration and underwent repeat bursa aspiration. No organisms were isolated or reported on Gram staining on any of the aspirate samples. Two aspirates were reported positive for calcium pyrophosphate dihydrate crystals. No patient had monosodium urate crystals. All patients had resolution of swelling and symptoms without the development of postaspiration infection., Conclusions: This study demonstrates limited clinical utility of routine microbiologic analysis (cell count, microbiologic, and crystal evaluation) of fluid aspirate from clinically diagnosed aseptic OB. Although 7% of fluid aspirates were positive for calcium pyrophosphate dihydrate crystals, it did not change the overall treatment., (© 2022 The Author(s).)

»: For shoulder arthroplasty, regional anesthesia is safer when compared with general anesthesia. There is insufficient evidence to demonstrate the superiority of regional anesthesia with respect to pulmonary complications and hospital length of stay., »: Infiltration of the shoulder with local anesthetics offers no additional benefits compared with single-shot or continuous brachial plexus blocks for shoulder arthroplasty., »: There is high-quality evidence (Level I) demonstrating lower pain scores and lower perioperative opioid requirements after a continuous peripheral nerve block compared with a single-shot nerve block. However, catheter dislodgment and logistical issues with catheter insertion are impediments to the widespread usage of a continuous nerve block with an indwelling catheter., »: Liposomal bupivacaine is comparable with non-liposomal local anesthetic agents with respect to pain relief, the opioid-sparing effect, and adverse effects in the first 48 hours after total shoulder arthroplasty., »: Perioperative dexamethasone administration improves postoperative pain control, decreases perioperative opioid requirements, and reduces postoperative nausea., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSREV/A780)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Biomaterial-associated infections (BAI), which are predominantly caused by Staphylococcus epidermidis, are a significant problem in modern medicine. Biofilm formation is considered the pivotal element in the pathogenesis, but in previous mouse studies we retrieved S. epidermidis from peri-implant tissue. To assess the kinetics and generality of tissue colonization, we investigated BAI using two S. epidermidis strains, two biomaterials, and two mouse strains. With small inocula all implants were culture negative, whereas surrounding tissues were positive. When higher doses were used, tissues were culture positive more often than implants, with higher numbers of CFU. This was true for the different biomaterials tested, for both S. epidermidis strains, at different times, and for both mouse strains. S. epidermidis colocalized with host cells at a distance that was >10 cell layers from the biomaterial-tissue interface. We concluded that in mouse experimental BAI S. epidermidis peri-implant tissue colonization is more important than biofilm formation.

Major predisposing conditions for infective endocarditis (IE) are the presence of a cardiac platelet-fibrin vegetation and of circulating bacteria with relatively low susceptibility to microbicidal activity of blood platelets. The influence of proinflammatory conditions on development of IE is unknown. We studied the effects of the presence of a catheter, inserted to induce platelet-fibrin vegetations, and of the proinflammatory cytokine interleukin-1alpha in rabbit experimental IE. Leaving the catheter in place after challenge with viridans streptococci predisposed for experimental IE. IE susceptibility rapidly decreased between 0 to 6 h after catheter removal. The catheter did not predispose for IE by providing a site for bacterial adherence, as almost all explanted catheters were culture negative. To mimic the proinflammatory influence of the catheter, rabbits were injected with interleukin-1alpha at 24 h after catheter removal and at 0, 1, and 3 h before bacterial challenge. Interleukin-1alpha injected 3 h prior to challenge significantly increased IE incidence due to a platelet releasate-susceptible Streptococcus oralis strain, with rapidly increasing numbers of bacteria within the vegetations. IE due to the Streptococcus sanguis strain less susceptible to platelet releasate was not enhanced. We conclude that proinflammatory stimuli, either a catheter or interleukin-1alpha, enhanced susceptibility to IE due to the platelet releasate-susceptible S. oralis. As with rabbits, temporary intravascular proinflammatory conditions may predispose for IE in humans at risk for this serious infection.

In order to come to a reliable evaluation of the effectiveness of the chosen vaccination policy regarding meningococcal disease, the completeness of registrations on meningococcal disease in the Netherlands was estimated with the capture-recapture method. Data over 1993-1998 were collected from (A) mandatory notifications (n = 2926); (B) hospital registration (n = 3968); (C) laboratory surveillance (n = 3484). As the standard capture-recapture method does not take into account false positive diagnoses, we developed a model to adjust for the lack of specificity of our sources. We estimated that 1363 cases were not registered in any of the three sources in the period of study. The completeness of the three sources was therefore estimated at 49% for source A, 67% for source B and 58% for source C. After adjustment for false positive diagnoses, the completeness of source A, B, and C was estimated as 52%, 70% and 62%, respectively. The capture-recapture methods offer an attractive approach to estimate the completeness of surveillance sources and hence contribute to a more accurate estimate of the disease burden under study. However, the method does not account for higher-order interactions or presence of false positive diagnoses. Being aware of these limitations, the capture-recapture method still elucidates the (in)completeness of sources and gives a rough estimate of this (in)completeness. This makes a more accurate monitoring of disease incidence possible and hence attributes to a more reliable foundation for the design and evaluation of health interventions such as vaccination programs.

Steam sterilization is the most common method of sterilization used in hospitals and by companies sterilizing for hospitals. This study validated 197 steam sterilizers with respect to technical condition, various production processes and routine control tests, according to the European norms and standards for steam sterilization. Overall, only 40% of the validated steam sterilizers met the norms and standards. We recommend that adequate measures need to be taken, based on the comments in the validation reports, in order to guarantee the sterility of processed medical items.

A 12-year-old Dutch boy was admitted because of severe neurotrauma after a traffic accident. On day 33 after admission, a Plasmodium falciparum infection was found in a routine blood smear. Most probably he was infected by blood of a patient next to him, a girl with severe malaria tropica. The genotype of the P. falciparum in both patients was identical.

The opacity (Opa) proteins of Neisseria meningitidis are outer membrane proteins involved in adhesion and invasion of host epithelial cells and are therefore expected to play an important role in colonisation of the nasopharynx. The majority of meningococcal Opa proteins bind to members of the CEACAM receptor family, such as CEA. Blocking of the Opa-CEACAM interaction by mucosal anti-Opa antibodies could thus constitute an important protective mechanism for novel meningococcal vaccines. In this study we analysed the specific anti-Opa antibody responses after intranasal immunisation of mice with liposomes containing purified and native OpaB (recognising the CEA receptor) and OpaJ (no affinity for CEA) proteins. These antigens were combined with or without one of three different adjuvants, i.e. purified meningococcal LPS, monophosphoryl lipid A (MPL) or the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB). After intranasal immunisation with any of these formulations, anti-Opa IgA antibodies were found in nasal lavages and in some cases anti-Opa IgA and IgG antibodies were also found in lung lavages. With OpaJ but not OpaB, significant bactericidal serum titres were obtained. Of the different adjuvants used, meningococcal LPS gave the strongest overall immune response. Non-adjuvated liposomal Opa formulations were poorly immunogenic. No differences were found between the immune response in transgenic mice expressing the CEA-receptor and non-transgenic mice, showing that the CEA-Opa interaction does not influence the antibody response.

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.

Clusters are recognized when meningococcal cases of the same phenotypic strain (markers: serogroup, serotype, and subtype) occur in spatial and temporal proximity. The incidence of such clusters was compared to the incidence that would be expected by chance by using space-time nearest-neighbor analysis of 4,887 confirmed invasive meningococcal cases identified in the 9-year surveillance period 1993-2001 in the Netherlands. Clustering beyond chance only occurred among the closest neighboring cases (comparable to secondary cases) and was small (3.1%, 95% confidence interval 2.1%-4.1%).

To study the effect of initial colonisation on Helicobacter pylori gene expression, altered H. pylori gene transcription during co-culture with human gastric epithelial cells was determined. Therefore, an insertion library of H. pylori with random chromosomal fusions to a promoterless cat gene was grown in the presence of HM02 gastric epithelial cells and varying levels of chloramphenicol. One H. pylori transformant was chloramphenicol-resistant in the presence, but chloramphenicol-susceptible in the absence of gastric epithelial HM02 cells. This transformant had the promoterless cat gene inserted into the HP0887 gene, which encodes the vacuolating cytotoxin VacA, an important virulence factor of H. pylori. Reverse transcriptase polymerase chain reaction on cDNA of this transformant confirmed vacA upregulation near HM02 cells. These results show the applicability of this technique to study H. pylori gene regulation in its natural environment.

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.

Tuberculosis remains one of the leading infectious causes of death worldwide. The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health threat. Resistance to isoniazid (INH) is the most prevalent form of resistance in M. tuberculosis and is mainly caused by mutations in the catalase peroxidase gene (katG). Among high-level INH-resistant isolates (MIC > or = 2), 89% are associated with a mutation at codon 315 of katG. There is a need to develop rapid diagnostic tests to permit appropriate antibiotic treatment and to improve clinical management. Therefore, a single-tube real-time PCR, using a novel kind of probe (3'-minor groove binder-DNA probe), was developed to detect either the wild-type or the mutant codon directly in Ziehl-Neelsen-positive sputum samples. The detection limit of the assay for purified DNA was 5 fg per well (one mycobacterial genome), and with spiked sputum samples, it was 20 copies per well, corresponding to 10(3) mycobacteria per ml of sputum. Sputum samples from 20 patients living in Kazakhstan or Moldova and infected with monodrug- or multidrug-resistant M. tuberculosis and 20 sputum samples from patients infected with INH-susceptible M. tuberculosis were tested. The sensitivities and specificities of the probes were 70 and 94% for the wild-type probe and 82 and 100% for the mutant probe. Binding to either probe was nonambiguous. This real-time PCR allows the rapid identification of a mutant katG allele and can easily be implemented in a clinical microbiology laboratory.

Background: Selective decontamination of the digestive tract (SDD) is an infection-prevention regimen used in critically ill patients. We assessed the effects of SDD on intensive-care-unit (ICU) and hospital mortality, and on the acquisition of resistant bacteria in adult patients admitted to intensive care., Methods: We did a prospective, controlled, randomised, unblinded clinical trial. 934 patients admitted to a surgical and medical ICU were randomly assigned oral and enteral polymyxin E, tobramycin, and amphotericin B combined with an initial 4-day course of intravenous cefotaxime (SDD group n=466), or standard treatment (controls n=468). Primary endpoints were ICU and hospital mortality and the acquisition of resistant bacteria., Findings: In the SDD group 69 (15%) patients died in the ICU compared with 107 (23%) in the control group (p=0.002). Hospital mortality was lower in the SDD groups than in the control group (113 [24%] vs 146 [31%], p=0.02). During their stay in intensive care, colonisation with gram-negative bacteria resistant to ceftazidime, ciprofloxacin, imipenem, polymyxin E, or tobramycin occurred in 61 (16%) of 378 SDD patients and in 104 (26%) of 395 patients in the control group (p=0.001). Colonisation with vancomycin-resistant enterococcus occurred in five (1%) SDD patients and in four (1%) controls (p=1.0). No patient in either group was colonised with meticillin-resistant Staphylococcus aureus., Interpretation: In a setting with low prevalence of vancomycin-resistant enterococcus and meticillin-resistant S aureus, SDD can decrease ICU and hospital mortality and colonisation with resistant gram-negative aerobic bacteria.

Diagnostic potential of the Chlamydia trachomatis ligase chain reaction system (LCx) to assess the presence of C. trachomatis in urine and semen specimens was evaluated. Paired urine and semen specimens from 153 asymptomatic male partners of subfertile couples attending our Center for Reproductive Medicine were examined by LCx. As controls, 19 semen samples from four donors who were participating in the programme for artificial insemination were used. Of these, 12 samples had previously been shown to be C. trachomatis-positive by an in-house PCR. C. trachomatis was detected by LCx in seven of 153 (5 %) urine samples. None of the 153 semen samples tested positive by LCx. Also, none of the 12 C. trachomatis-containing control semen samples were positive by LCx. By in-house PCR, seven urine specimens and two of 153 (1 %) semen samples tested positive. The corresponding urine samples of these male partners were also C. trachomatis-positive, as well as the 12 C. trachomatis-containing samples from donors. In conclusion, LCx is not sensitive enough to assess the presence of C. trachomatis in semen specimens; therefore, this method is not recommended to routinely screen semen specimens from donors who participate in programmes for artificial insemination or male partners of subfertile couples for C. trachomatis.

Objective: To describe the initial effects of the large-scale vaccination campaign in June-July of 2002 (1-5- and 15-18-year-olds) and September-November of 2002 (6-14-year-olds) on the incidence of group-C meningococcal disease in the Netherlands., Design: Descriptive., Method: The incidence of meningococcal disease and the serogroup distribution were determined on the basis of the patient data associated with isolates of Neisseria meningitidis that were sent to the Netherlands Reference Laboratory for Bacterial Meningitis during the period from 1 January 1999 to 31 January 2003., Results: The highest monthly incidence of serogroup-C disease was reported in January-April 2002 (2.2-3.1/100,000), after which the incidence remained more or less unchanged after September at a level of 0.1-0.4/100,000. The incidence of meningococcal-C disease was 73% lower in August-October 2002 compared with the same period in 2001. In the same months, the incidence of meningococcal-C disease for the 0, 1-5, 6-14, 15-18 and > 18 year-olds was 49%, 80%, 89%, 89% and 42%, respectively, lower than in the same months in the previous year. The percentage of meningococcal disease caused by serogroup-C fluctuated from 35 to 49% in January-August 2002 and decreased to 9-19% in September-December 2002. There was also a decrease in the unvaccinated age groups., Conclusion: The vaccination campaign led almost immediately to a sharp decrease in the number of patients with meningococcal serogroup-C disease.

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Background: Investigation of the rate of active conversion of flucytosine to fluorouracil by microorganisms in the intestinal microflora., Methods: Active conversion of flucytosine was investigated using viable and nonviable Escherichia coli at different flucytosine concentrations. Additionally, flucytosine conversion was studied in fecal specimens from 3 neutropenic patients at the start of the antimicrobial/antifungal prophylaxis (C/A regimen) and 1 week later., Results: Flucytosine levels decreased by an average of 72, 71 and 72% flucytosine after incubation for 48 h of 10(10) viable E. coli /ml suspension in broth containing 13, 130 and 1300 mg/l flucytosine, respectively. The decreasing flucytosine levels corresponded approximately to an identical increase in fluorouracil levels. Also, a 44% decrease of flucytosine levels occurred when nonviable E. coli were used, indicating that bacterial viability is not necessary for this conversion. When fecal specimens of 2 patients were investigated prior to the C/A regimen, significant flucytosine conversion occurred, whereas this conversion was not observed in the corresponding fecal specimens after 1 week of C/A regimen., Conclusion: These in vitro experiments showed that extensive flucytosine conversion can occur in the human intestinal microflora by E. coli. Consequently, fluorouracil exposure and fluorouracil-related toxicity may occur in the flucytosine-treated patient., (Copyright 2003 S. Karger AG, Basel)

During a prospective study of 8 months duration conducted in the Department of Internal Medicine and the Department of Pulmonary Diseases of the Academic Medical Centre, Amsterdam, Gram stainings of sputa and urine were performed for all patients whose clinical symptoms indicated an acute urinary tract infection or pulmonary infection. On the test request, the physician reported which antibiotic treatment he would prescribe if a microscopic examination was not available. The results of the Gram stain were discussed by the microbiologist with the physician, and the antibiotic therapy recommended by the microbiologist was recorded. This recommendation was compared with the antibiotic prescription noted in the patient record 1 day later. Two days after the results of final cultures and susceptibility tests became available, the patient record was again investigated for changes in the antibiotic regimen. Of 57 urine samples and 103 sputa, 27 and 85, respectively, were derived from patients with an infection of the urinary tract or respiratory tract. The results of the Gram stain confirmed the physician's suspicion 91% of the time for urinary tract infections and 81% of the time for pulmonary infections. In 67% of the patients with suspected lower respiratory tract infections and in 58% of patients with suspected urinary tract infections, the antibiotic treatment recommended on the basis of the results of the Gram stain differed significantly from the antibiotic treatment that the physician would have prescribed had a microscopic examination not been performed. The microbiologist's advice on antibiotic treatment was followed in 79% of the cases of respiratory tract infection and in 65% of the cases of urinary tract infection. The antibiotic treatment was adjusted to the final results of culture and antimicrobial susceptibility testing in 54% of the urinary tract infections and in 31% of the respiratory tract infections. The results indicate that the examination of sputa and urine in patients suspected to have an infection of the respiratory tract or urinary tract influences the antibiotic choice considerably.

An outbreak of 7 cases of group C meningococcal disease occurred during the last week of July and the first week of August 2001 in the southwestern part of The Netherlands. Characterization of the 7 patients' isolates by various typing methods showed that the isolates were identical, except for the expression of PorA. Isolates from 5 patients were PorA deficient. These results show that transmission of PorA-deficient meningococci occurs and that PorA-deficient meningococci can cause invasive disease. PorA-based meningococcal vaccines may provide limited protection.

Stimulated neutrophils release a variety of antimicrobial peptides, including neutrophil defensins (HNP1-4). We have previously reported that neutrophil defensins enhanced the adherence of Haemophilus influenzae and Neisseria meningitidis to cultured respiratory epithelial cells. In this study, the effect of defensins on the adherence of H. influenzae and N. meningitidis lipooligosaccharide (LOS) mutants to epithelial cells was tested. Neutrophil defensins enhanced the adherence of the oligosaccharide mutants of H. influenzae and N. meningitidis, whilst the adherence of the lipid A mutants B29 of H. influenzae and lpxL1 and lpxL2 of N. meningitidis was not or only moderately stimulated by neutrophil defensins. The adherence of the N. meningitidis LOS negative mutant lpxA was not enhanced by defensins. These findings suggested that the secondary fatty acids of lipid A were involved in the defensin-enhanced adherence. LOS from strain H44/76 or HNP-LOS complexes did not affect or stimulate the adherence of N. meningitidis, although the defensin-enhanced adherence is specific for certain bacterial species having LOS in their outer membrane. These results indicated that LOS is involved in the defensin-enhanced adherence. However, the mechanism by which defensins and LOS interact with epithelial cells to promote bacterial adherence remains to be resolved.

The sensitivity and specificity of the Cell-Dyn 4000 hematology analyzer in the diagnosis of imported malaria were studied with samples from patients in an academic hospital setting. The performance of the Cell-Dyn 4000 hematology analyzer was compared with that of conventional diagnostic methods for malaria. The Cell-Dyn 4000 hematology analyzer detected hemozoin-containing depolarizing monocytes in 29 of 58 patients with malaria and 2 of 55 patients without malaria. The presence or absence of depolarizing monocytes in patients with malaria was related to duration of symptoms before presentation for malaria analysis. A second parameter, pseudoreticulocytosis due to nuclear material of intraerythrocytic malaria parasites, was detected by the Cell-Dyn 4000 hematology analyzer almost exclusively in Plasmodium falciparum malaria patients with parasitemia levels of >/=0.5%. Attention to these abnormalities in medical centers without tropical disease expertise may decrease a delay in the diagnosis of malaria.

Adults without neurologic sequelae after bacterial meningitis are supposed to live without restrictions. Neuropsychological outcome was assessed in 51 adults from a prospective cohort with good recovery, defined as Glasgow Outcome Scale score 5, after pneumococcal or meningococcal meningitis. Patients who recovered well after pneumococcal meningitis showed cognitive slowness (P=.001). A cognitive disorder was found in 27% of these patients. Patients who previously had meningococcal meningitis were not significantly different from control subjects. Scores on general health and quality of life questionnaires revealed lower scores for patients with meningitis, which were related to cognitive slowing (R, -0.46 to -0.38). In conclusion, adults surviving pneumococcal meningitis were at significant risk of neuropsychological abnormalities, even if they were clinically well recovered.

In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the "gold standard" in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used.

Encapsulated meningococci are complement sensitive only in the presence of bactericidal antibodies by yet-unexplored mechanisms. The objective of this study was to investigate the involvement of major bacterial surface constituents on complement activation and membrane attack complex (MAC) formation on serogroup B meningococci in the presence or absence of antibody-dependent serum bactericidal activity (SBA). The strains used were the encapsulated H44/76, five of its variants differing in capsulation and expression of the class 1 porin (PorA), and its lipopolysaccharide (LPS)-deficient isogenic mutant (LPS(-)) pLAK33. Two normal sera, one with high SBA (SBA(+)) and one with no bactericidal activity (SBA(-)) against H44/76 as well as an a-gamma-globulinemic serum were used for sensibilization of the bacteria. C3b and iC3b deposition on H44/76, its unencapsulated variant v24, and pLAK33 was similar in SBA(+) and SBA(-) serum, and no difference was present between the strains. MAC deposition on H44/76 was higher in SBA(+) serum than in SBA(-) serum and the a-gamma-globulinemic serum. The amounts of C3b on H44/76, v24, and pLAK33 in the a-gamma-globulinemic serum were also not different, indicating immunoglobulin G (IgG)- and LPS-independent complement activation. H44/76 PorA(+) and its PorA(-) variant and the v24 PorA(+) and its PorA(-) variant incubated in SBA(-) serum induced comparable amounts of MAC, despite their different serum sensitivities. Complement formation on the surface of the bacteria occurred almost exclusively via the classical pathway, but the considerable amounts of Bb measured in the serum indicated alternative pathway activation in the fluid phase. We conclude that complement deposition on meningococci is, for the most part, independent of classical pathway IgG and is not influenced by the presence of PorA or LPS on the meningococcal surface. Addition of an anti-PorA chimeric antibody to the nonbactericidal normal serum, while promoting a dose-related bacterial lysis, did not influence the amounts of C3b, iC3b, and MAC formed on the bacterial surface. These findings support the hypothesis that proper MAC insertion rather than the quantity of MAC formed on the bacterial surface is of importance for efficient lysis of meningococci.

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.

Two hundred and seven cases of listeria meningitis that occurred in The Netherlands over 20 years were reviewed to study associations between Listeria monocytogenes serotype, age, underlying disease, and outcome. The mean annual incidence per 100,000 population was 0.12 in 1981-90, decreasing to 0.07 in 1991-5. Underlying disease was present in 50% of non-neonatal patients, most often haematological malignancy (15%) and the use of immunosuppressive therapy (14%). The meningitis-related case fatality rate was 16%; a significantly higher rate was associated with the presence of underlying disease (30%) or age > or = 70 years (29%). Serotype 4b was most frequent (65%) and L. monocytogenes types 1/2a, 1/2b, or 1/2c (30% of cases) were significantly more often isolated from non-neonatal patients with underlying disease, suggesting a higher virulence of listerial serotype 4b.

Chart review of 41 adult patients with group A streptococcal (GAS) meningitis in The Netherlands revealed that this is a community-acquired disease and occurs mainly in patients with predisposing factors: of the 41 patients in this case series, 24 (60%) of 40 evaluable patients had otitis or sinusitis. Fever and neck stiffness were the most common clinical manifestations of disease, but, in addition, high rates of seizures (12 [32%] of 38 patients), focal neurological findings (13 [36%] of 36 patients), and hyponatremia (20 [58%] of 35 patients) were found. In contrast with data from the literature that describes 27 adult cases, we found that GAS meningitis is a fulminant disease with a mortality rate of 27% (10 of 37 patients), and that neurological sequelae occur in 36% (12 of 38) of surviving patients.

In The Netherlands, national guidelines for the treatment of adult patients with bacterial meningitis were introduced in October 1997. In 1998 we began a prospective, nationwide study to evaluate the compliance with these consensus-based guidelines. In addition, we evaluated whether the recommended initial treatment provides adequate microbiological coverage. From October 1998 to January 2000, 365 adults with bacterial meningitis were identified using information from The Netherlands Reference Laboratory for Bacterial Meningitis; 263 patients were classified into four categories depending on patient's age and underlying health status. In the first category, patients 16-60 years without risk factors, Neisseria meningitidis was the most common pathogen (53%); 62 of 127 patients (49%) received treatment in compliance with the guidelines. In the second and third categories, patients >60 years without risk factors and those with risk factors independently of age, Streptococcus pneumoniae caused 61% and 58% of cases, respectively. Compliance in these categories was about 17%. Overall, 33% of patients received treatment in compliance with the guidelines. The microbiological coverage of patients treated in compliance and not in compliance with the guidelines was 98% and 93%, respectively. In conclusion, 1 year after national consensus-based guidelines for the initial treatment of adult patient with bacterial meningitis were introduced in The Netherlands, only one-third of Dutch physicians were adhering to the guidelines. The microbiological coverage for the patients who were treated in compliance with the guidelines was almost complete (98%).

The aim of this study is to investigate whether fluorouracil (5-FU) could be responsible for bone-marrow depression occurring in fluorocytosine (5-FC) treated patients. Six 5-FC treated patients were included in this pilot study. Toxicity was monitored by means of thrombocyte and leucocyte counts. 5-FC and 5-FU serum levels were measured using a high-performance liquid chromatography (HPLC) assay that allows simultaneous determination of both compounds. The amounts of 5-FU in the 34 available serum samples remained below the limit of quantitation (< 0.05 mg/L), whereas 5-FC levels could be detected in all samples. Instead, low levels of the 5-FU catabolite alpha-fluoro-beta-alanine (FBAL) were detected in several of the investigated serum samples. In case of three patients thrombocyte counts remained within the normal range during 5-FC treatment, whereas one patient developed thrombocytopenia (50 x 10(9) thrombocytes/L) during therapy. Furthermore, one patient developed leucocytopenia (2.6 x 10(9) leucocytes/L) during 5-FC therapy, whereas the remaining five patients were suffering from leucocytosis prior to 5-FC therapy. In conclusion, we found nondetectable 5-FU serum concentrations (< 0.05 mg/L) in ICU patients treated with intravenous 5-FC, making it unlikely that 5-FC-associated toxicity results from 5-FU exposure in patients receiving intravenous 5-FC therapy. These findings may be explained by the fact that our patients received 5-FC intravenously instead of orally, therefore not allowing active conversion of 5-FC to 5-FU by the human intestinal microflora.

Of 96 Helicobacter pylori isolates from patients in Shanghai and Guangzhou, China, 5 had the A2143G 23S rRNA mutation as determined by primer-mismatch PCR and were resistant to clarithromycin by the E-test. The remaining isolates were primer-mismatch PCR negative and susceptible to clarithromycin. The conclusion is that the prevalence of clarithromycin-resistant H. pylori isolates among these Chinese patients is 5%.

The population structure of Neisseria meningitidis is supposedly epidemic according to. The model predicts that linkage disequilibrium in N. meningitidis populations is only temporary and arises due to the outgrowth of highly successful clonal genotypes from an essentially sexual population. These clones should disappear after a few years because of frequent recombination. In contrast, multilocus enzyme electrophoresis (MLEE) data had previously been interpreted as showing that serogroup A meningococci are truly clonal and possess only limited genetic variability (Wang et al., 1992). The two interpretations are contradictory. In order to elucidate the true population structure of serogroup A meningococci, we analyzed data for a representative group of 84 serogroup A isolates obtained by MLEE, random amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). Analysis of linkage disequilibrium and bootstrap analyses of cluster analysis showed a strongly structured population with highly significant linkage disequilibrium. This was not due to the overrepresentation of certain genotypes, in contrast to the expectations for an epidemic population. The analyses identify two main clades, within each of which linkage disequilibrium was also highly significant, thus, excluding a cryptic speciation model. These observations support a population structure based on clonal evolution, in which clones are much more stable than expected for epidemic clonality. We propose that serogroup A meningococci may possess a different population structure from other serogroups of Neisseria meningitidis.

To study the role of platelet microbicidal activity in host defense against infective endocarditis (IE) due to viridans streptococci (VS), the susceptibility to platelet releasate of blood and oral VS isolates from patients with and without IE was compared. The influence of neutralization of platelet microbicidal activity was studied in 2 experimental IE models. Resistance to platelet releasate was more prevalent among VS from blood of patients with IE than from blood of bacteremic patients without IE and among oral VS isolates. Serum from rabbits vaccinated with human platelet sonicate supernatants neutralized human and rabbit platelet-released microbicidal activity and had antibodies recognizing microbicidal proteins thrombocidin-1 and -2 and other human platelet proteins. In the 2 rabbit IE models, vaccination increased the susceptibility to experimental IE due to platelet releasate-susceptible VS. Thus, platelet-released microbicidal activity is an important host defense factor against IE due to VS.

Mycoplasma pneumoniae strains traditionally are divided into two types, based on sequence variation in the P1 gene. Recently, however, we have identified 8 P1 subtypes by restriction fragment length polymorphism analysis. In the present study the P1 gene sequences of three P1 type 1 and two P1 type 2 M. pneumoniae strains were analyzed. A new P1 gene sequence in a type 1 strain with partial similarity to a recently reported variable region in the P1 gene of an M. pneumoniae type 2 strain (T. Kenri, R. Taniguchi, Y. Sasaki, N. Okazaki, M. Narita, K. Izumikawa, M. Umetsu, and T.Sasaki, Infect. Immun. 67:4557-4562, 1999) was identified. In addition, the P1 gene of the type 1 strain contained another region with nucleotide polymorphisms identical to a stretch in the P1 gene of one of our type 2 strains. These findings indicate that recombination between sequences specific for P1 type 1 and type 2 had occurred and that P1 type 1 and type 2 hybrid sequences can be present within the P1 gene of an individual strain. Identical or nearly identical variable P1 gene sequences were present in several repetitive regions outside the P1 gene locus in the genome of M. pneumoniae strain M129, implying recombination as a mechanism for generation of the P1 gene variation. Additionally, in the P1 gene sequences of four of the five strains studied, single-nucleotide polymorphisms different from the previously reported P1 type 1 and 2 characteristic sequences were identified. The polymorphic sites are candidate targets for genotyping of M. pneumoniae by direct sequencing of amplicons from clinical specimens.

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.

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

Of the Helicobacter pylori populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous H. pylori population, resistant H. pylori colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible H. pylori colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous H. pylori populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary H. pylori population is used to test for clarithromycin susceptibility.

Instruments used by dentists are often sterilized with steam sterilization. At this moment three types of processes are described in the European norms: Type N, S and B. According to the norms type N can be used for non-wrapped non-hollow instruments and non-porous instruments, type S can be used for instruments specified by the manufacturer and type B can be used for wrapped, hollow and porous instruments. The principles on which the sterilizors are based are described. It is concluded that before purchasing the sterilizor the method of working and instruments to be sterilized must specified. In most cases a type B process is preferable in order to gaurantee to be certain to have an effective and reproducible process.

The VMP-like sequence (vls) locus that consists of one expressed vlsE gene and 15 silent vls cassettes has been described in Borrelia burgdorferi sensu stricto B31. In the present study, the vls locus from a Borrelia garinii isolate A87SA was analyzed. DNA fragments that contained three complete and five partial vls cassettes were cloned and sequenced. Pulsed-field gel electrophoresis (PFGE) analysis and Southern hybridization of the PFGE blot indicated that the vls locus of B. garinii A87SA, consisting of at least eight vls cassettes, was located on a 21-kb linear plasmid. The size of the three complete vls cassettes varied from 573 to 612 bp. They had 93.8-94.3% identity at the nucleotide level and 84.9-87.3% amino acid identity. The amino acid sequences of the three vls cassettes of B. garinii A87SA exhibited 45.9-50.8% identity to the VlsE sequence of B. burgdorferi B31, and 30.0-33.8% identity to the VMP17 sequence of B. hermsii HS1. Homologues of the vls locus of B. garinii were detected by dot blot hybridization among 24 of the 30 (80.0%) isolates representing four B. burgdorferi sensu lato species distributed widely in Europe. Our findings indicate that B. garinii might possess a similar vls structure to that described in B. burgdorferi sensu stricto. The highly conserved nature of the vls locus among various B. burgdorferi sensu lato species suggests that it may be important in the physiology and pathogenesis of Lyme disease spirochetes.

Aims: The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested., Methods and Results: E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics., Conclusion: Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors., Significance and Impact of the Study: The elucidation of the mechanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies.

Reactive arthritis (ReA) after infections with various gram-negative bacteria is strongly associated with the MHC class I molecule HLA-B27. It is supposed that the B27 molecule itself plays a role in the pathogenesis of ReA by presenting antigenic peptides to cytotoxic T lymphocytes. The peptide repertoires presented by Salmonella-, Shigella- and non-infected cells were compared to identify such peptides. From the peptides isolated from the B27 molecules of these cells, profiles were generated by reversed-phase chromatography and peaks present in the profiles from infected cells but not in profiles from non-infected cells were studied for their peptide compositions. Some sequences with identity to those in human histone H3, human ribosomal protein S17 and the heavy chain of HLA-B27 itself were detected only in profiles from infected cells. All peptides identified from infected cells contained the B*2705 peptide-binding motif. The data suggest that HLA-B27-positive cells infected with ReA-inducing bacteria show an increased presentation of certain self-peptides. There was no evidence for altered peptide-binding specificity of B27 after infection. However, the interpretations were hampered by the variation in peptide presentation between different experiments.

A mutation (CCG-->CTG [Arg-->Leu]) in codon 463 of katG (catalase peroxidase) of Mycobacterium tuberculosis has been found in isoniazid (INH)-resistant strains. A PCR restriction endonuclease analysis to detect this mutation was applied to 395 M. tuberculosis isolates from patients in The Netherlands. The proportion of isolates with a detectable mutation was 32% (32 out of 100) and 29% (85 out of 295) among INH-susceptible isolates and INH-resistant or -intermediate isolates, respectively. Sequencing of five INH-susceptible isolates with such mutations showed that all five had the Arg463Leu mutation. We conclude that the Arg463Leu mutation of katG of M. tuberculosis is not a reliable indicator of INH resistance.

Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between lineage III strains and non-lineage III strains were identified using representational difference analysis. Thus, a 1.8-kb locus that is specific for lineage III meningococci was identified. The locus contains three open reading frames encoding the NmeSI restriction-modification system. The methyltransferase gene was cloned and expressed in Escherichia coli. Site AGTACT was found to be modified by the enzyme. In conclusion, lineage III strains differ from endemic strains by the presence of a specific restriction-modification system. This restriction-modification system may contribute to the clonal and hypervirulent character of lineage III strains by influencing horizontal gene transfer and transcription.

During a 30-month prospective study in The Netherlands, the distribution of Mycoplasma pneumoniae and respiratory viruses among 1172 patients with acute respiratory infection (ARI) who were treated in the outpatient general practitioner setting was studied. M. pneumoniae, as detected by polymerase chain reaction analysis, was present in 39 (3.3%) patients. The infection rate was similar in all age groups. Nose and throat samples collected from 79 household contacts of M. pneumoniae-positive index patients revealed M. pneumoniae in 12 (15%) cases. The frequency of M. pneumoniae among household contacts of index patients treated with appropriate antibiotics and untreated index patients was similar. Nine of the 12 M. pneumoniae-positive household contacts were <16 years old (P=.02), and 4 (44%) of them did not develop ARI. Apparently, children are a relevant reservoir for M. pneumoniae.

In this study the decontamination procedures of laryngoscopes in Dutch hospitals are described, based on a structured telephone questionnaire. There were substantial differences between decontamination procedures in Dutch hospitals and the standards of the APIC (Association of Professionals in Infection Control and Epidemiology), CDC (Centers of Disease Control) and ASA (American Society of Anesthesiology) were met in full in 19.4% of the hospitals. The standards of manual decontamination, used in 78% of the 139 hospitals, were particularly disappointing; manual cleaning was considered inadequate in 22.9% of these hospitals and manual disinfection did not meet the standards of the APIC, CDC or ASA in any of these hospitals. Decontamination by instrument cleaning machines as a standard procedure was used in 30 (22%) hospitals. In three of these hospitals the blades were subsequently sterilized. We suggest adherence to the infection control guidelines of the CDC, APIC and ASA, until the safety of less conservative infection control practices are demonstrated.