Faecal carriage, risk factors, acquisition and persistence of ESBL-producing Enterobacteriaceae in dogs and cats and co-carriage with humans belonging to the same household.

BACKGROUND: ESBL-producing Enterobacteriaceae (ESBL-E) are observed in many reservoirs. Pets might play an important role in the dissemination of ESBL-E to humans since they live closely together. OBJECTIVES: To identify prevalence, risk factors, molecular characteristics, persistence and acquisition of ESBL-E in dogs and cats, and co-carriage in human-pet pairs belonging to the same household. METHODS: In a nationwide study, one person per household was randomly invited to complete a questionnaire and to submit a faecal sample. Dog and cat owners were invited to also submit a faecal sample from their pet. Repeated sampling after 1 and 6 months was performed in a subset. ESBL-E were obtained through selective culture and characterized by WGS. Logistic regression analyses and random forest models were performed to identify risk factors. RESULTS: The prevalence of ESBL-E carriage in these cohorts was 3.8% (95% CI: 2.7%-5.4%) for human participants (n=550), 10.7% (95% CI: 8.3%-13.7%) for dogs (n=555) and 1.4% (95% CI: 0.5%-3.8%) for cats (n=285). Among animals, blaCTX-M-1 was most abundant, followed by blaCTX-M-15. In dogs, persistence of carriage was 57.1% at 1 month and 42.9% at 6 months. Eating raw meat [OR: 8.8, 95% CI: 4.7-16.4; population attributable risk (PAR): 46.5%, 95% CI: 41.3%-49.3%] and dry food (OR: 0.2, 95% CI: 0.1-0.5; PAR: 56.5%, 95% CI: 33.2%-66.6%) were predictors for ESBL-E carriage in dogs. Human-dog co-carriage was demonstrated in five households. Human-cat co-carriage was not observed. CONCLUSIONS: ESBL-E prevalence was higher in dogs than in humans and lowest in cats. The main risk factor for ESBL-E carriage was eating raw meat. Co-carriage in dogs and household members was uncommon.

Dynamics of colistin and tobramycin resistance among Enterobacter cloacae during prolonged use of selective decontamination of the digestive tract.

Background: A high prevalence of colistin resistance among E. cloacae isolates in two intensive care units (ICU) (of 16 and 6 beds) using selective digestive decontamination (SDD) since 1990 instigated a retrospective and prospective investigation to quantify the role of clonal transmission. SDD is topical application of colistin and tobramycin and systemic use of cefotaxime during the first days of ICU-admission. Methods: Multi-resistant E. cloacae (MREb) was defined as ESBL production and/or tobramycin non-susceptibility and/or colistin non-susceptibility. Incidence of acquisition and prevalence of carriage with MREb was determined from microbiological culture results. Results: Colistin-resistant E. cloacae was first detected in November 2009 and carriage was demonstrated in 141 patients until October 2014. Mean incidence of MREb acquisition was 4.61 and 1.86 per 1000 days at risk in ICUs 1 and 2, respectively, and the mean monthly prevalence of MREb in both ICUs was 7.0 and 3.1%, respectively, without a discernible trend in time. Conversion rates from carriage of colistin-susceptible to resistant E. cloacae were 0.20 and 0.13 per 1000 patient days, respectively. Whole genome sequencing of 149 isolates revealed eight clusters, with the number of SNPs of the largest two clusters ranging between 0 and 116 for cluster 1 (n = 49 isolates), and 0 and 27 for cluster 2 (n = 36 isolates), among isolates derived between 2009 and 2014. Conclusions: This study demonstrates a stable low-level endemicity of MREb in two Dutch ICUs with prolonged use of SDD, which was characterized by the persistent presence of two clusters, suggesting incidental clonal transmission.

Quantifying within-household transmission of extended-spectrum ß-lactamase-producing bacteria.

OBJECTIVES: Patients can acquire extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during hospitalization, and colonized patients may transmit these bacteria after discharge, most likely to household contacts. In this study, ESBL transmission was quantified in households. METHODS: Faecal samples were longitudinally collected from hospitalized patients colonized with ESBL-producing bacteria and from their household members during hospitalization of the index patient and at 3, 6, 12 and 18 months. A mathematical household model was developed, which allowed for person-to-person transmission, acquisition from other sources (background transmission), and losing carriage. Next, a deterministic population model with a household structure was created, informed by parameter values found in the household model. RESULTS: In all, 74 index patients and 84 household members were included. In more than half of the household members ESBL-producing bacteria were demonstrated at some time during follow up. Person-to-person transmission occurred at a rate of 0.0053/colonized person/day (0.0025-0.011), background transmission at 0.00015/day (95% CI 0.00002-0.00039), and decolonization at 0.0026/day (0.0016-0.0040) for index patients and 0.0090/day (0.0046-0.018) for household members. The estimated probability of transmission from an index patient to a household contact was 67% and 37% vice versa. CONCLUSION: There is frequent transmission of ESBL-producing bacteria in households, which may contribute to the observed endemicity of ESBL carriage in the Netherlands. However, the population model suggests that there is not a single dominant acquisition route in the community.

Carriage of extended-spectrum ß-lactamases in pig farmers is associated with occurrence in pigs.

Livestock may serve as a reservoir for extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE). The objectives of this study were to determine the prevalence of carriage with ESBL-PE in pig farmers, family members and employees, and its association with carriage in pigs. Rectal swabs were taken from 2388 pigs (398 pooled samples) on 40 pig farms and faecal samples were obtained from 142 humans living or working on 34 of these farms. Presence of ESBL-PE was determined by selective plating (agar). ESBL genes were analysed by PCR or microarray analysis, and gene sequencing. Genotypes and plasmids were determined by multilocus sequence typing and PCR-based replicon typing for selected isolates. ESBL genes were detected in Escherichia coli from eight humans (6%) (blaCTX-M-1, n = 6; blaTEM-52, n = 1 and blaCTX-M-14, n = 1) on six farms. In 157 pig isolates (107 pooled samples) on 18 farms (45%) ESBL genes were detected (blaCTX-M-1, n = 12; blaTEM-52, n = 6; and blaCTX-M-14, n = 3). Human and pig isolates within the same farm harboured similar ESBL gene types and had identical sequence and plasmid types on two farms (e.g. E. coli ST-453, blaCTX-M-1, IncI1), suggesting clonal transmission. For the remaining farms, sequence types, but not plasmid types, differed. Human ESBL carriage was associated with average number of hours working on the farm per week (OR = 1.04, 95% CI 1.02-1.06) and presence of ESBLs in pigs (OR = 12.5, 95% CI 1.4-111.7). Daily exposure to pigs carrying ESBL-PE is associated with ESBL carriage in humans.

Predicting carriage with extended-spectrum beta-lactamase-producing bacteria at hospital admission: a cross-sectional study.

The prevalence of patients colonized with extended-spectrum beta-lactamase (ESBL)-producing bacteria increases, especially in long-term-care facilities (LTCFs). Identification of ESBL carriers at hospital admission is relevant for infection control measures and antibiotic therapy for nosocomial infections. We aimed to develop a prediction rule for ESBL carriage at hospital admission for patients admitted from home and LTCFs, and to quantify incidences of nosocomial infections caused by ESBL-producing bacteria. The ESBL-carrier status was determined of patients admitted from LTCFs and from home settings in four hospitals in the Netherlands using perianal swabs obtained within 48 hours of admission. Risk factors for ESBL carriage were assessed. Infections caused by ESBL-producing bacteria were identified retrospectively. Among 1351 patients, 111 (8.2%) were ESBL carriers at admission: 50/579 (8.6%) admitted from LTCFs and 61/772 (7.9%) from home settings (p 0.63). Previous ESBL carriage and previous hospital admission were risk factors for ESBL carriage in multivariable analysis. The area under the curve of the receiver operating characteristic curve of the model was 0.64 (95% CI 0.58-0.71). Presence of ≥1 risk factor (n = 803; 59%) had sensitivity of 72%. Incidences of nosocomial infections caused by ESBL-producing bacteria were 45.5/10,000 and 2.1/10,000 admission days for ESBL carriers and non-carriers, respectively (p <0.05). In conclusion, prevalence of ESBL carriage at hospital admission was 8.2%, and was comparable among patients admitted from LTCF and home. A clinically useful prediction rule for ESBL carriage at admission could not be developed. The absolute incidence of nosocomial infections by ESBL-producing bacteria was low, but higher among patients carrying ESBL-producing bacteria at the time of hospital admission.

A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid, dipicolinic acid and temocillin.

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 µg), temocillin (30 µg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96-100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae.

Evaluation of the Oxoid Brilliance™ CRE Agar for the detection of carbapenemase-producing Enterobacteriaceae.

The adequate detection of carbapenemase-producing Enterobacteriaceae (CPE) is essential for adequate antibiotic therapy and for infection control purposes, especially in an outbreak setting. Selective agars play an important role in the detection of CPE. The Oxoid Brilliance™ CRE Agar (Thermo Fisher Scientific) was evaluated for the detection of CPE using 255 non-repetitive Enterobacteriaceae isolates, including 95 CPE (36 KPC, 4 KPC plus VIM, 4 NDM, 6 GIM, 20 VIM, and 25 OXA-48-producing isolates). The sensitivity of the CRE agar for the detection of CPE was 94 % (89/95), but differed per carbapenemase gene (100 % for KPC, NDM, and GIM, 90 % for VIM, and 84 % for OXA-48-producing isolates). The specificity of the CRE agar was 71 %, due to the growth of AmpC- and/or ESBL-producing isolates. The CRE agar is a sensitive tool for the detection of KPC and metallo-carbapenemase-producing Enterobacteriaceae, although the detection of OXA-48 producers is less optimal. The relatively low specificity requires confirmation of carbapenemase production for isolates recovered from the CRE agar.

Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum beta-lactamases.

The concurrent presence of bla CTX-M-1 and bla TEM-52 genes on similar plasmids of Escherichia coli isolated from poultry, chicken meat and humans supports the occurrence of food-borne transmission of extended-spectrum beta-lactamase (ESBL) genes. ESBL-producing E. coli (ESBL-E. coli) are most frequently detected in hospitalised patients and are known to spread in healthcare settings. We hypothesised that poultry-associated (PA) ESBL genes are predominant in the community, where acquisition is fuelled by food contamination, whereas non-PA ESBL genes are predominant in hospitals, with acquisition fuelled by cross-transmission. Then, differences in antimicrobial selective pressure in hospitals and poultry would create differences in co-resistance between PA and non-PA ESBL-E. coli. We, therefore, determined the prevalence and co-resistance of PA and non-PA ESBL-E. coli in community-acquired and nosocomial urinary tract infections in humans and bla CTX-M-1 and bla TEM-52 isolates from poultry. A total of 134 human ESBL-E. coli urine isolates were included in this study. Isolates containing bla CTX-M-1 or bla TEM-52 were considered to be PA, with the remainder being non-PA. Also, 72 poultry ESBL-E. coli were included. Minimum inhibitory concentration (MIC) values were determined by broth microdilution. The prevalence of PA ESBL genes in isolates obtained in general practice and hospitals was 28 % versus 30 % (n.s.). Human PA ESBL-E. coli were more frequently susceptible to ciprofloxacin (51 % vs. 25 %; p = 0.0056), gentamicin (86 % vs. 63 %; p = .0.0082), tobramycin (91 % vs. 34 %; p = 0.0001) and amikacin (98 % vs. 67 %; p = 0.0001) compared to human non-PA ESBL-E. coli. PA ESBL-E. coli are not more prevalent in community acquired than nosocomial urine samples, but are more often susceptible to ciprofloxacin and aminoglycosides than non-PA ESBL-E. coli. This does not support the existence of different reservoirs of ESBL genes.

Multi-centre evaluation of a phenotypic extended spectrum ß-lactamase detection guideline in the routine setting.

This study aimed to evaluate the routine setting performance of a guideline for phenotypic detection of extended spectrum ß-lactamases (ESBLs) in Enterobacteriaceae, recommending ESBL confirmation with Etest or combination disc for isolates with a positive ESBL screen test (i.e. cefotaxime and/or ceftazidime MIC >1 mg/L or an automated system ESBL warning). Twenty laboratories submitted 443 Enterobacteriaceae with a positive ESBL screen test and their confirmation test result (74%Escherichia coli, 12%Enterobacter cloacae, 8%Klebsiella pneumoniae, 3%Proteus mirabilis, 2%Klebsiella oxytoca). Presence of ESBL genes was used as reference test. Accuracy of local phenotypic ESBL detection was 88%. The positive predictive value (PPV) of local screen tests was 70%, and differed per method (Vitek-2: 69%, Phoenix: 68%, disc diffusion: 92%), and species (95%K. pneumoniae-27%K. oxytoca). A low PPV (3%) was observed for isolates with automated system alarm but third-generation cephalosporin MICs <2 mg/L. Local ESBL confirmation had a PPV and negative predictive value (NPV) of 93% and 90%, respectively. Compared with centrally performed confirmation tests, 7% of local tests were misinterpreted. Combination disc was more specific than Etest (91% versus 61%). Confirmation tests were not reliable for P. mirabilis and K. oxytoca (PPV 33% and 38%, respectively, although NPVs were 100%). In conclusion, performance of Etests could be enhanced by education of technicians to improve their interpretation, by genotypic ESBL confirmation of P. mirabilis and K. oxytoca isolates with positive phenotypic ESBL confirmation, and by interpreting isolates with a positive ESBL alarm but an MIC <2 mg/L for cefotaxime and ceftazidime as ESBL-negative.

Evaluation of a commercial microarray as a confirmation test for the presence of extended-spectrum ß-lactamases in isolates from the routine clinical setting.

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105).

Rapid detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis.

OBJECTIVES: Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS: Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS: The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS: This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

The echinocandin caspofungin impairs the innate immune mechanism against Candida parapsilosis.

Since caspofungin inhibits fungal cell wall beta-glucan synthesis and the fungal cell wall plays an important role in the recognition of Candida by phagocytic cells, we studied phagocytosis in the presence of caspofungin. The aim of this work was to investigate the effect of pre-treatment of Candida parapsilosis with caspofungin on phagocytic mechanisms (opsonisation, oxidative burst, phagocytosis and killing). C. parapsilosis grown in the presence of caspofungin at concentrations above the minimal inhibitory concentration (MIC) were more difficult to opsonise and to phagocytose. C. parapsilosis exposed to any concentration of caspofungin below and above the MIC was more difficult to kill. Caspofungin-treated C. parapsilosis impaired the oxidative burst. Overall, it appears that caspofungin treatment of C. parapsilosis alters the capacity of polymorphonuclear leukocytes to phagocytose and delays killing of the organism. This may allow C. parapsilosis to persist in tissues.

Identification of genotypically diverse Cryptococcus neoformans and Cryptococcus gattii isolates by Luminex xMAP technology.

A Luminex suspension array, which had been developed for identification of Cryptococcus neoformans and Cryptococcus gattii isolates, was tested by genotyping a set of 58 mostly clinical isolates. All genotypes of C. neoformans and C. gattii were included. In addition, cerebrospinal fluid (CSF) obtained from patients with cryptococcal meningitis was used to investigate the feasibility of the technique for identification of the infecting strain. The suspension array correctly identified haploid isolates in all cases. Furthermore, hybrid isolates possessing two alleles of the Luminex probe region could be identified as hybrids. In CSF specimens, the genotype of the cryptococcal strains responsible for infection could be identified after optimization of the PCR conditions. However, further optimization of the DNA extraction protocol is needed to enhance the usability of the method in clinical practice.

Antibody neutralization of vascular endothelial growth factor (VEGF) fails to attenuate vascular permeability and brain edema in experimental pneumococcal meningitis.

To determine the contribution of vascular endothelial growth factor (VEGF) to cerebral edema formation in bacterial meningitis, we used a VEGF neutralizing antibody to block VEGF in rabbits, following induction of meningitis by intracisternal inoculation with 10(9) heat-killed pneumococci. At 8 h, cerebrospinal fluid (CSF) VEGF was significantly elevated in infected untreated animals, and correlated with CSF white blood cell (WBC) count (r=0.56, P=0.004), and brain water content (r=0.42, P=0.04). Blocking of VEGF did not attenuate brain edema, blood-brain barrier disruption, or CSF pleocytosis. The functional role of VEGF in the pathophysiology of BM remains elusive.

Potent inhibition of neutrophil migration by cryptococcal mannoprotein-4-induced desensitization.

Cryptococcal capsular Ags induce the production of proinflammatory cytokines in patients with cryptococcal meningitis. Despite this, their cerebrospinal fluid typically contains few neutrophils. Capsular glucuronoxylomannan is generally considered to mediate the inhibition of neutrophil extravasation. In the current study, culture supernatant harvested from the nonglucuronoxylomannan-producing strain CAP67 was found to be as potent as supernatant from wild-type strains in preventing migration. We identified capsular mannoprotein (MP)-4 as the causative agent. Purified MP-4 inhibited migration of neutrophils toward platelet-activating factor, IL-8, and fMLP, probably via a mechanism involving chemoattractant receptor cross-desensitization, as suggested by its direct chemotactic activity. Supporting this hypothesis, MP-4 elicited Ca(2+) transients that were inhibited by preincubation with either fMLP, IL-8, or C5a, but not platelet-activating factor, and vice versa. Moreover, MP-4 strongly decreased the neutrophil surface expression of L-selectin and induced shedding of TNF receptors p55/p75, whereas CD11b/18 increased. Finally, MP-4 was clearly detectable in both serum and cerebrospinal fluid of patients suffering from cryptococcal meningitis. These findings identify MP-4 as a novel capsular Ag prematurely activating neutrophils and desensitizing them toward a chemoattractant challenge.

Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader.

Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).

Pulmonary surfactant protein A binds to Cryptococcus neoformans without promoting phagocytosis.

BACKGROUND: Evidence is accumulating that the alveolar collecting surfactant protein A (SP-A) plays an important role in the first line of defence against infiltrating pathogenic micro-organisms and viruses. The ability of SP-A to facilitate the binding and uptake of acapsular Cryptococcus neoformans by monocyte-derived macrophages, human alveolar macrophages, monocytes and polymorphonuclear leucocytes was investigated. MATERIALS AND METHODS: Binding, competition and phagocytosis experiments were performed using a flow cytometry technique. RESULTS: SP-A bound to both the acapsular and the encapsulated form of C. neoformans in a concentration-dependent manner. SP-A showed a threefold better binding to the acapsular yeast: this binding was partly calcium dependent and could be inhibited by mannose (ID50 = 3 mmol L-1) and glucose (ID50 = 2.1 mmol L-1) but not by galactose (ID50 = 391 mmol L-1). SP-A did not function as an opsonin in phagocytosis of acapsular C. neoformans for any of the phagocytes studied. CONCLUSION: Our results indicate that SP-A binds in a concentration-dependent manner to both encapsulated and acapsular C. neoformans. Despite SP-A binding to the acapsular C. neoformans, phagocytosis by various phagocytes was not enhanced.

Induction of TNF-alpha in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein.

We have shown previously that specific receptors on PBMCs and a serum factor other than Ab and complement are involved in the TNF-alpha response to cryptococcal mannoprotein (MP2). To characterize the mechanism of MP2 recognition by PBMCs, 10(6) PBMCs were incubated with 25 microg of FITC-labeled MP2 in 10% normal human serum (1 h). The cells were analyzed by flow cytometry. FITC-MP2 binding was CaCl2 and temperature dependent and was enhanced by prestimulating PBMCs with unlabeled MP2. Binding to PBMCs was specific, since unlabeled MP and mannan produced dose-dependent inhibition. Beta-Glucan laminarin produced background inhibition. mAbs against CD14, CD11b, and CD18 did not prevent FITC-MP2 binding to PBMCs, implying that these receptors are not involved in MP2 recognition by PBMCs. mAb against CD14 blocked (>90%) MP2-induced TNF-alpha release by PBMCs, while mAbs against CD11b/CD18 caused no inhibition. Removal of human mannose binding protein (hMBP) by preincubation of serum with a specific mAb abrogated TNF-alpha induction by MP2 and strongly inhibited its binding to PBMCs. Recombinant hMBP enhanced TNF-alpha induction by MP2 as well as binding of FITC-MP2 to PBMCs. In addition, incubation of serum with MP2-coated beads and analysis by SDS-PAGE resulted in the detection of a protein of approximately 33/34 kDa that could be partially removed by preincubating the serum with hMBP mAb. We conclude that hMBP is involved in the binding of MP2 to PBMCs and the release of TNF-alpha.

Mould-devouring mites differ in guanine excretion from dust-eating Acari, a possible error source in mite allergen exposure studies.

BACKGROUND AND OBJECTIVE: Measurement of guanine in dust proved a good assessment of mite allergen exposure. METHODS: Exposure to mite allergens may lead to atopic inflictions. In a semi-natural test system the development of Dermatophagoides pteronyssinus (Trouessart) and Glycyphagus domesticus (De Geer), and the presence of their guanine excretion, was examined in a dust-soiled and mouldy environment. Mites were counted after heat-escape, and guanine was detected by means of capillary zone electrophoresis. For each species, 50 mites randomly taken, were inoculated on soiled test-surfaces of 10 x 10 cm. Rough wooden board, gypsum board, tufted carpet, and a self-made mattress representing wall surfaces and home-textiles, respectively, were used. Eight weeks after inoculation with mites only, the surfaces were all mould ridden, and mite and guanine measurements were taken. The Spearman rank correlation test and the Mann-Whitney U-test were used in statistical analysis. The confidence limit was set at 1%. RESULTS: Among the various test-surfaces, no differences were found regarding total mite numbers and amount of guanine present (P > 0.01). For the dust-eating mite D. pteronyssinus, total mite numbers correlated with the amount of guanine present (P = 0.002) on all inoculated surfaces, indicating feeding on the protein-rich dust. For the mould devouring mite G. domesticus, however, no such correlation was found (P = 0.72). Apparently, they mainly consumed fungal carbohydrates during this experiment. CONCLUSION: The allergological relevance of storage mites has been under discussion for the last 25 years. In humid homes, these mites will feed almost exclusively on fungi and may produce allergenic or irritating substances different from those arising on protein-rich laboratory media used in allergen extract production or present in carpets, bedding and furniture.

Cryptococcus neoformans and cryptococcal glucuronoxylomannan, galactoxylomannan, and mannoprotein induce different levels of tumor necrosis factor alpha in human peripheral blood mononuclear cells.

Tumor necrosis factor alpha (TNF-alpha) release by peripheral blood mononuclear cells (PBMC) during disseminated infection by Cryptococcus neoformans may initiate and amplify the immune response of the host, leading to elimination of the fungus. The ability to induce TNF-alpha in PBMC by four clinical strains of C. neoformans, a laboratory strain (NIH 37), and the purified cryptococcal components glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MP1 and MP2) were investigated under different opsonic conditions. In the absence of serum, the levels of TNF-alpha induced by all strains and cryptococcal components were not above background levels. Normal human serum (NHS) enhanced TNF-alpha induction by whole cryptococci and the different cryptococcal components, with MP2 being the most potent TNF-alpha inducer. Inactivation of complement (HI NHS) almost abrogated the ability of whole cryptococci and the GXMs to induce TNF-alpha. In contrast, when MP1, MP2, and GalXM were incubated with HI NHS, 48, 71, and 44%, respectively, of the original TNF-alpha levels remained. MPs incubated with heat-inactivated immunoglobulin G (IgG)-depleted serum still induced 50% of the levels of TNF-alpha induced by components incubated with HI NHS. Both these sera contained the same very low levels of anti-MP IgG antibodies, indicating the opsonic effect of a heat-stable factor other than antibody. Two anti-CD14 monoclonal antibodies (60BCA and 3C10) inhibited the production of TNF-alpha induced by MP2. The results indicate that (i) induction of TNF-alpha by C. neoformans and GXMs strongly depends on complement, (ii) MP1 and MP2 induction of TNF-alpha is facilitated by a heat-stable serum factor other than Ig, and (iii) CD14 may be involved in the induction of TNF-alpha by MP2.