Basophil activation test with progressively less heated forms of egg distinguishes egg allergic from tolerant children.

BACKGROUND AND OBJECTIVE: Diagnosis of egg allergy through basophil activation testing (BAT) has been mainly performed with an egg white extract or individual egg allergens rather than clinically more representative whole-egg extracts. Impact of heating on whole-egg extract allergenicity remains unassessed.Validating BAT with gradually less heated whole-egg extracts in egg allergy diagnosis and as tolerance marker. METHODS: CD63-based BAT was performed with five progressively less heated extracts from cake, hard-boiled egg, omelet, soft-boiled, and raw egg in 10 egg allergic (EA), 10 complete egg tolerant (ET) and 12 non-egg-sensitized non-allergic (NEA) children. Cutoffs and diagnostic accuracy measures were established through ROC analysis. Changes in basophil response were assessed in 12 baked egg tolerant children undergoing an 8-month gradual egg reintroduction protocol with BAT and oral food challenges prior to each reintroduction step. RESULTS: Basophil responses to all egg extracts were increased in EA, but not in ET and NEA children. Responses decreased progressively with more heated egg extracts. Compared to ET children, EA children showed higher basophil sensitivity for all egg extracts. Negative BAT responses predicted clinical tolerance with a 90-100% sensitivity, 100% specificity, and false positive rate of 2.78%. In comparison, egg sIgE's (<0.35 kUA/L) had a lower specificity of 50-78% with a false positive rate of 40%. Basophil reactivity and sensitivity tended to decrease in baked egg tolerant children undergoing gradual egg reintroduction, concurrent with tolerance development. CONCLUSION: BAT with progressively less heated egg preparations is a sensitive and highly specific tool to discriminate EA from ET children.

Diagnosing COVID-19; towards a feasible COVID-19 rule-out protocol.

INTRODUCTION: We present the results of the COVID-19 rule-out protocol at Ghent University Hospital, a step-wise testing approach which included repeat NFS SARS-CoV-2 rRT-PCR, respiratory multiplex RT-PCR, low-dose chest CT and bronchoscopy with BAL to confirm or rule-out SARS-CoV-2 infection in patients admitted with symptoms suggestive of COVID-19. RESULTS: Between 19 March 2020 and 30 April 2020, 455 non-critically ill patients with symptoms suspect for COVID-19 were admitted. The initial NFS for SARS-CoV-2 rRT-PCR yielded 66.9%, the second NFS 25.4% and bronchoscopy with BAL 5.9% of total COVID-19 diagnoses. In the BAL fluid, other respiratory pathogens were detected in 65% (13/20) of the COVID-19 negative patients and only in 1/7 COVID-19 positive patients. Retrospective antibody testing at the time around BAL sampling showed a positive IgA or IgG in 42.9 % of the COVID-19 positive and 10.5% of the COVID-19 negative group. Follow-up serology showed 100% COVID-19 positivity in the COVID-19 positive group and 100% IgG negativity in the COVID-19 negative group. CONCLUSION: In our experience, bronchoscopy with BAL can have an added value to rule-in or rule-out COVID-19 in patients with clinical and radiographical high-likelihood of COVID-19 and repeated negative NFS testing. Furthermore, culture and respiratory multiplex PCR on BAL fluid can aid to identify alternative microbial etiological agents in this group. Retrospective analysis of antibody development in this selected group of patients suggests that the implementation of serological assays in the routine testing protocol will decrease the need for invasive procedures like bronchoscopy.

Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload.

Toilet drain water as a potential source of hospital room-to-room transmission of carbapenemase-producing Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) have rapidly emerged in Europe, being responsible for nosocomial outbreaks. AIM: Following an outbreak in the burn unit of Ghent University Hospital, we investigated whether CPE can spread between toilets through drain water and therefrom be transmitted to patients. METHODS: In 2017, the burn centre of our hospital experienced an outbreak of OXA-48-producing Klebsiella pneumoniae that affected five patients staying in three different rooms. Environmental samples were collected from the sink, shower, shower stretcher, hand rail of the bed, nursing carts, toilets, and drain water to explore a common source. Whole-genome sequencing and phylogenetic analysis was performed on K. pneumoniae outbreak isolates and two random K. pneumoniae isolates. FINDINGS: OXA-48-producing K. pneumoniae was detected in toilet water in four out of six rooms and drain water between two rooms. The strain persisted in two out of six rooms after two months of daily disinfection with bleach. All outbreak isolates belonged to sequence type (ST) 15 and showed isogenicity (<15 allele differences). This suggests that the strain may have spread between rooms by drain water. Unexpectedly, one random isolate obtained from a patient who became colonized while residing at the geriatric ward clustered with the outbreak isolates, suggesting the outbreak to be larger than expected. Daily application of bleach tended to be superior to acetic acid to disinfect toilet water; however, disinfection did not completely prevent the presence of carbapenemase-producing K. pneumoniae in toilet water. CONCLUSION: Toilet drain water may be a potential source of hospital room-to-room transmission of carbapenemase-producing K. pneumoniae.

Identifying a consensus sample type to test for Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis and human papillomavirus.

OBJECTIVES: Sexually transmitted infections (STIs) are a global cause of acute illness. Early detection plays a crucial role in interrupting transmission and preventing complications. However, the accessibility of STI testing is curbed by the lack of an overall preferred sample type. By means of a prospective study in female sex workers (FSW), we compared the sensitivity of samples from different anatomical sites in detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium and human papillomavirus. Besides, we documented the prevalence of each STI in this high-risk population. METHODS: We selected 303 FSW and tested them for each STI by nucleic acid amplification testing on two vaginal and cervical swabs from different manufacturers, cervical smear and first-void urine. The sensitivity of each sample type was compared for each infectious agent in order to identify a consensus sample type. RESULTS: Vaginal swabs were superior to all other sample types, with an overall sensitivity of 86%. The sensitivity was the lowest for first-void urine, detecting only 63% of positive cases. The prevalence was 3.3% (10/299) for Neisseria gonorrhoeae; 9.0% (27/299) for Chlamydia trachomatis; 7.4% (22/298) for Trichomonas vaginalis; 10.8% (32/296) for Mycoplasma genitalium and 55.6% (158/284) for human papillomavirus. CONCLUSIONS: When testing for STIs, vaginal swabs are the sample of choice and first-void urine should be avoided. Designating (self-sampled) vaginal swabs as a consensus sample type enables harmonization of STI testing and extension of testing to large numbers of unscreened females.

Women with symptoms of a urinary tract infection but a negative urine culture: PCR-based quantification of Escherichia coli suggests infection in most cases.

OBJECTIVES: Our objective was to examine whether or not women with symptoms of a urinary tract infection but with a negative culture (20%-30%) do have an infection. METHODS: We performed quantitative PCR (qPCR) for Escherichia coli and Staphylococcus saprophyticus, on top of a standard culture, in urine samples from 220 women with dysuria and/or frequency and/or urgency and from 86 women without symptoms. For symptomatic women, qPCR was also carried out for four sexually transmitted agents. RESULTS: In the symptomatic group, 80.9% (178/220) of the urine cultures were positive for any uropathogen and 95.9% (211/220) were E. coli qPCR-positive. For the control group, cultures for E. coli and E. coli qPCR were positive in, respectively, 10.5% (9/86) and 11.6% (10/86). In the symptomatic group, qPCR yielded 19 positive samples for S. saprophyticus qPCR, one positive sample for Mycoplasma genitalium and one for Trichomonas vaginalis. CONCLUSIONS: These findings suggest that almost all women with typical urinary complaints and a negative culture still have an infection with E. coli.

The resident microflora of voided midstream urine of healthy controls: standard versus expanded urine culture protocols.

The workup and interpretation of urine cultures is not always clear-cut, especially for midstream samples contaminated with commensals. Standard urine culture (SUC) protocols are designed in favor of growth of uropathogens at the expense of commensals. In selected clinical situations, however, it is essential to trace fastidious or new uropathogens by expanding the urine culture conditions (EUC). The aim of our study was to map the microflora in midstream urine specimens from healthy controls by means of EUC, in view of the interpretation of bacterial culture results in symptomatic patients. Midstream urine specimens from 101 healthy controls (86 females and 15 males) were examined using both SUC and EUC. Whilst 73 % of samples examined by SUC showed no growth at 103 colony-forming units (CFU)/mL, 91 % of samples examined by EUC grew bacterial species in large numbers (≥104 CFU/mL). Asymptomatic bacteriuria, as defined by the European guidelines for urinalysis, was detected in six samples with both protocols. EUC revealed 98 different species, mostly Lactobacillus, Staphylococcus, Streptococcus, and Corynebacterium. None of the samples grew Staphylococcus saprophyticus, Corynebacterium urealyticum, or Aerococcus urinae. Samples from females contained higher bacterial loads and showed higher bacterial diversity compared to males. Midstream urine of healthy controls contains large communities of living bacteria that comprise a resident microflora, only revealed by EUC. Hence, the use of EUC instead of SUC in a routine setting would result in more sensitive but less specific results, requiring critical interpretation. In our view, EUC should be reserved for limited indications.

Evaluation of Copan FLOQSwab for the molecular detection of Chlamydia trachomatis by Abbott RealTime CT PCR.

OBJECTIVES: We evaluated Copan FLOQSwabs next to Abbott swabs for the detection of Chlamydia trachomatis (CT) by Abbott RealTime PCR. METHODS: We collected 1062 paired swabs from female sex workers. The study was divided in two arms, according to the order of swab collection. RESULTS: If the Abbott swab was collected first, 501 couples were concordant and two discordant (Abbott negative and Copan positive). If the Copan swab was collected first, 537 couples were concordant and 10 discordant (eight Abbott negative and Copan positive and two Abbott positive and Copan negative). All discordant samples contained low levels of C. trachomatis. Technical issues lead to retesting of 64 Copan and 21 Abbott swabs. CONCLUSION: Our results show that Copan FLOQSwabs can be used interchangeably with Abbott swabs. While appearing to have an advantage in detecting more positive samples, the use of Copan swabs led to a higher retesting rate due to technical errors.

Evaluation of the BD BACTEC FX blood volume monitoring system as a continuous quality improvement measure.

The yield of blood cultures is proportional to the volume of blood cultured. We evaluated an automatic blood volume monitoring system, recently developed by Becton Dickinson within its BACTEC EpiCenter module, that calculates mean volumes of negative aerobic bottles and generates boxplots and histograms. First, we evaluated the filling degree of 339 aerobic glass blood cultures by calculating the weight-based volume for each bottle. A substantial amount of the bottles (48.3%) were inadequately filled. Evaluation of the accuracy of the monitoring system showed a mean bias of -1.4 mL (-15.4%). Additional evaluation, using the amended software on 287 aerobic blood culture bottles, resulted in an acceptable mean deviation of -0.3 mL (-3.3%). The new software version was also tested on 200 of the recently introduced plastic bottles, which will replace the glass bottles in the near future, showing a mean deviation of +2.8 mL (+26.7%). In conclusion, the mean calculated volumes can be used for the training of a single phlebotomist. However, filling problems appear to be masked when using them for phlebotomist groups or on wards. Here, visual interpretation of boxplots and histograms can serve as a useful tool to observe the spread of the filling degrees and to develop a continuous improvement program. Re-adjustment of the software has proven to be necessary for use with plastic bottles. Due to our findings, BD has developed further adjustments to the software for validated use with plastic bottles, which will be released soon.

Direct susceptibility testing by disk diffusion on clinical samples: a rapid and accurate tool for antibiotic stewardship.

We compared the accuracy of direct susceptibility testing (DST) with conventional antimicrobial susceptibility testing (AST), both using disk diffusion, on clinical samples. A total of 123 clinical samples (respiratory tract samples, urine, vaginal and abdominal abscess discharges, bile fluid and a haematoma punctate) were selected on various indications; direct inoculation on Mueller-Hinton agar and antibiotic paper disks were applied. In parallel, standard culture, identification and AST on the colonies grown overnight was executed. Both AST and DST were interpreted after identification of the isolates. The results from both AST and DST for 11 antibiotics tested on 97 samples with Gram-negative rods showed 93.4 % total agreement, 1.6 % minor discordances, 4.6 % major discordances and 0.4 % very major discordances. Analysing the discordant results, DST predominantly resulted in more resistant isolates than AST. This was mostly due to the presence of resistant mutants or an additional isolate. The remaining discordances were seen for isolates with inhibition zones close to the clinical breakpoint. For the 26 samples yielding staphylococci, a total agreement of 100 % was observed for the nine antibiotics tested. Overall, the highest percentage of discordant results occurred for the ß-lactam antibiotics amoxicillin-clavulanate (13.4 %) and cefuroxime (12.4 %). When used selectively and interpreted carefully, DST on clinical samples is potentially very useful in the management of critically ill patients, as the time to results is shortened by approximately 24 h. However, we recommend to communicate results with reservations and confirm by conventional AST.

Breakthrough Saprochaete capitata infections in patients receiving echinocandins: case report and review of the literature.

We report a rare case of a fatal Saprochaete capitata breakthrough infection in a patient with acute myeloid leukemia receiving empirical caspofungin therapy. S. capitata is an uncommon, yet emerging cause of invasive infections, especially in patients with haematological malignancies. Blood cultures from our patient yielded S. capitata which was found to be resistant, in vitro, to caspofungin. We consecutively reviewed all published cases of breakthrough infections caused by S. capitata in patients receiving echinocandins. S. capitata should be considered in those patients who remain febrile or who develop invasive mould infections while under echinocandin therapy.

Cyclosporin A increases IFN-gamma production by T cells when co-stimulated through CD28.

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-gamma production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-gamma (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with costimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-gamma production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4+ cells was responsible for the enhancement of IFN-gamma production in the presence of CsA. In conclusion, IFN-gamma production by CD28-co-stimulated CD4+ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma.

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3+ T cells, with a clear predominance of CD4- CD8+ over CD4+ CD8- T cells, and a marked population of CD4+ CD8+ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3+ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3+ CD4+ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3+ CD8+ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3+ CD4+ TIL in RCC have normal functional capacities, whereas the proportionally major CD3+ CD8+ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.

B7-CD28 interaction is a late acting co-stimulatory signal for human T cell responses.

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered as the most important co-stimulatory signal for T cell activation. APC in a resting condition express either no or only low levels of B7 molecules. These are up-regulated as a result of interactions with activated T cells, thus suggesting that B7-CD28 interaction is not required at initiation of T cell activation. To study this issue, we blocked B7-CD28 interaction at various time points after in vitro stimulation of peripheral blood T cells with allogeneic monocytes. Epstein-Barr virus-transformed B cells or soluble antigens. We observed that T cell proliferation and IL-2 production were inhibited by B7-blocking agents (CTLA-4-Ig or anti-B7 mAb) almost to the same degree when added either at initiation of culture or 24 h later. B7-blocking agents still resulted in significant inhibition of allogeneic T cell activation when added after 48 h. Furthermore, when CTLA-4-Ig was added at the start of an allogeneic T cell stimulation, addition of anti-CD28 mAb after 24 h of culture nearly fully restored T cell proliferation to control levels. Finally, we demonstrate that delayed addition of B7-blocking agents together with cyclosporin A 1 day after the onset of culture of T cells with allogeneic B cells is highly efficient to induce energy as evaluated by lack of proliferation, cytotoxic T lymphocyte reactivity and IFN-gamma or IL-5 production upon alloantigen rechallenge. Taken together, our data can explain why B7 expression on APC is not required at the time of initial APC-T cell contact, and suggest that the effect of the CD28 signal indeed consists in prolonging IL-2 production and amplifying T cell responses, rather than in providing a critical co-stimulatory signal at the time of initial TCR triggering.

Expression of B7-2 (CD86) molecules by Reed-Sternberg cells of Hodgkin's disease.

Ligation of CD28 on T cells with its natural ligands B7-1 (CD80) or B7-2 (CD86) provides a major costimulatory signal for T cells and is of potential importance for tumor rejection. We previously reported a strong expression of B7-1 on Reed-Sternberg cells and anaplastic large cell lymphoma cells. We report here our findings on B7-2 expression by malignant lymphomas (n = 70). B7-2 was present on the neoplastic cells of anaplastic large cell lymphoma in two of three cases studied, and on a subpopulation of the malignant cells in one out of four cases of follicular lymphoma. B7-2 was not expressed by the neoplastic cells of the other non-Hodgkin's lymphomas (n = 32), including T cell-rich B cell lymphoma. In contrast, Reed-Sternberg cells in lymph nodes affected by Hodgkin's disease are strongly positive for B7-2 (n = 31). Evidence for a functional correlate of this expression was obtained by our findings that the combination of anti-B7-1 and anti-B7-2 monoclonal antibodies was more effective than each separately in blocking allogeneic T cell activation (proliferation and cytokine secretion) by Hodgkin's disease-derived cell lines as stimulators. The possible role of B7-1 and B7-2 expression for the course and symptomatology of Hodgkin's disease is discussed.

Identification of an enriched CD4+ CD8alpha++ CD8beta+ T-cell subset among tumor-infiltrating lymphocytes in human renal cell carcinoma.

Three-color immunofluorescence flow-cytometric analysis of freshly isolated tumor-infiltrating lymphocytes (TIL) from patients with primary renal cell carcinoma (RCC) revealed a unique, not previously described TIL subset with a CD3+ CD4+ CD8alpha++ CD8beta+ phenotype. This subset represented at least 5% of CD3+ TIL in 15 of 21 patients with clear cell RCC, whereas it was not or only marginally represented in patients with papillary RCC or sarcomatoid RCC. In one-third of the patients with clear cell RCC, more than 20% of CD3+ TIL and in one patient more than half of the CD3+ TIL displayed this phenotype. The occurrence of this subset was not associated with pathological stage, tumor diameter, nuclear grade, cytogenetic abnormalities or vascular invasion in this patient cohort. When present, the CD3+ CD4+ CD8alpha++ CD8beta+ subset was detected in similar proportions in tumor tissue and tumor capsula, and it was also detected in adjacent non-tumoral renal tissue, albeit in much lower proportions. Despite strong cell surface expression of various activation markers (CD69, CD54 and HLA-DR), CD3+ CD4+ CD8alpha++ CD8beta+ cells displayed no ex vivo cytolytic activity in an anti-CD3-redirected cytotoxicity assay. In contrast with CD3+ CD4+ CD8- cells from the same tumor sample, they were markedly deficient in IL-2R alpha up-regulation following anti-CD3 triggering. The possibility that these cells represent either anergic cells or a highly specialized effector population with a discrete, as yet undescribed function is discussed.

Granulocyte-macrophage colony-stimulating factor down-regulates CD14 expression on monocytes.

CD14 is a differentiation-stage-linked glycosyl-phophatidyl-inositol-linked glycoprotein on human peripheral blood monocytes and tissue macrophages, which functions as a receptor for lipopolysaccharide. Here, the effects of granulocyte macrophage colony-stimulating factor (GM-CSF1 a cytokine with proliferation- and differentiation-inducing properties on myeloid lineage cells, were studied on CD14 expression by peripheral blood cells. GM-CSF down-regulated the membrane expression of CD14 on monocytes while it up-regulated expression on neutrophils. GM-CSF also decreased the spontaneous release of CD14 in monocyte culture supernatants. Down-regulation of CD14 expression and release was accompanied by a decrease in the mRNA transcript for CD14, suggesting that it most likely reflects an effect on the transcriptional level. The functional significance of this phenomenon, and its potential relation to the terminal differentiation of monocytes, are discussed.