VIDAS3® TB-IGRA assay: evaluation of performance characteristics in a predominantly low risk, low incidence population.

OBJECTIVES: To evaluate the analytical performance of the TB-IGRA® assay on the VIDAS3 platform (bioMérieux) when testing a predominantly low risk population in a low incidence area. RESULTS: Eighty-eight percent of the results were concordant between QuantiFERON®-TB Gold-Plus (QFT®-Plus, QIAGEN) and TB-IGRA®. All 12 of 99 (12.1%) discordant results were determined positive only with the TB-IGRA® assay. In 11 of 12 of these discordant cases, no explanation could be found in the medical record. Five of these discrepant results were probably caused by the use of contaminated stimulation reagents. The remaining 6 discrepant samples were also part of the reproducibility experiment and only 2 results were reproducible positive. Overall, in the reproducibility experiment 5 of 25 (20.0 %) results were not repeatable. CONCLUSIONS: the TB-IGRA® assay seems prone to contamination. Besides, we documented a reproducibility of only 80.0% with the TB-IGRA® assay.

Predictors and Dynamics of the Humoral and Cellular Immune Response to SARS-CoV-2 mRNA Vaccines in Hemodialysis Patients: A Multicenter Observational Study.

BACKGROUND: Preliminary evidence suggests patients on hemodialysis have a blunted early serological response to SARS-CoV-2 vaccination. Optimizing the vaccination strategy in this population requires a thorough understanding of predictors and dynamics of humoral and cellular immune responses to different SARS-CoV-2 vaccines. METHODS: This prospective multicenter study of 543 patients on hemodialysis and 75 healthy volunteers evaluated the immune responses at 4 or 5 weeks and 8 or 9 weeks after administration of the BNT162b2 or mRNA-1273 vaccine, respectively. We assessed anti-SARS-CoV-2 spike antibodies and T cell responses by IFN-γ secretion of peripheral blood lymphocytes upon SARS-CoV-2 glycoprotein stimulation (QuantiFERON assay) and evaluated potential predictors of the responses. RESULTS: Compared with healthy volunteers, patients on hemodialysis had an incomplete, delayed humoral immune response and a blunted cellular immune response. Geometric mean antibody titers at both time points were significantly greater in patients vaccinated with mRNA-1273 versus BNT162b2, and a larger proportion of them achieved the threshold of 4160 AU/ml, corresponding with high neutralizing antibody titers in vitro (53.6% versus 31.8% at 8 or 9 weeks, P <0.0001). Patients vaccinated with mRNA-1273 versus BNT162b2 exhibited significantly greater median QuantiFERON responses at both time points, and a larger proportion achieved the threshold of 0.15 IU/ml (64.4% versus 46.9% at 8 or 9 weeks, P <0.0001). Multivariate analysis identified COVID-19 experience, vaccine type, use of immunosuppressive drugs, serum albumin, lymphocyte count, hepatitis B vaccine nonresponder status, and dialysis vintage as independent predictors of the humoral and cellular responses. CONCLUSIONS: The mRNA-1273 vaccine's greater immunogenicity may be related to its higher mRNA dose. This suggests a high-dose vaccine might improve the impaired immune response to SARS-CoV-2 vaccination in patients on hemodialysis.

Ruminococcus gnavus bacteremia, an uncommon presentation of a common member of the human gut microbiota: case report and literature review.

Case report: We present a case of a 66-year-old female diagnosed with R. gnavus bacteremia associated with fecal peritonits secondary to small-bowel herniation and perforation. Identification  as R. gnavus was delayed because of absence of this species in the MALDI-TOF MS database (Vitek MS, bioMérieux). Identification was provided by 16S rRNA gene sequencing. Review: R. gnavus, a Gram-positive, strictly anaerobic bacterium, is a member of the human gut microbiota. Dysbiosis in the gut microbiota, with increased amounts of R. gnavus, has been described in inflammatory bowel disease. R. gnavus has only been reported occasionally as the cause of infections. Hence the potential pathogenicity is not yet fully recognized, and data regarding the antimicrobial susceptibility profile are rare. Identification of anaerobic bacteria such as R. gnavus is greatly accelerated  as a result of the introduction of MALDI-TOF MS. However, as illustrated in this case report, an extensive and up-to-date MALDI-TOF MS database is necessary for providing an accurate identification.

Nontuberculous mycobacteria among pulmonary tuberculosis patients: a retrospective Belgian multicenter study.

OBJECTIVES: Currently, there are no European data about the frequency and clinical significance of nontuberculous mycobacteria (NTM) grown from respiratory samples during the treatment of tuberculosis (TB). We determined the frequency and clinical significance of NTM isolated before or during pulmonary tuberculosis treatment in Belgian laboratories. METHODS: We conducted a nationwide retrospective multicenter cohort study on the co-isolation of TB and NTM in Belgium. Starting from laboratory data between 2006 and 2013, possible TB-NTM co-isolations were searched for. RESULTS: A total of 2569 unique culture-positive pulmonary tuberculosis cases were included in the study. Only 35 (1.4%) of these TB cases had an NTM co-isolated, and two of these 35 fulfilled the ATS criteria for NTM lung disease. CONCLUSION: A very low prevalence of 1.4% NTM co-isolations was found in Belgian patients with culture-proven pulmonary TB.

Nationwide Surveillance of Azole Resistance in Aspergillus Diseases.

Aspergillus disease affects a broad patient population, from patients with asthma to immunocompromised patients. Azole resistance has been increasingly reported in both clinical and environmental Aspergillus strains. The prevalence and clinical impact of azole resistance in different patient populations are currently unclear. This 1-year prospective multicenter cohort study aimed to provide detailed epidemiological data on Aspergillus resistance among patients with Aspergillus disease in Belgium. Isolates were prospectively collected in 18 hospitals (April 2011 to April 2012) for susceptibility testing. Clinical and treatment data were collected with a questionnaire. The outcome was evaluated to 1 year after a patient's inclusion. A total of 220 Aspergillus isolates from 182 patients were included. The underlying conditions included invasive aspergillosis (n = 122 patients), allergic bronchopulmonary aspergillosis (APBA) (n = 39 patients), chronic pulmonary aspergillosis (n = 10 patients), Aspergillus bronchitis (n = 7 patients), and aspergilloma (n = 5 patients). The overall azole resistance prevalence was 5.5% (95% confidence interval [CI] 2.8 to 10.2%) and was 7.0% (4/57; 95% CI, 2.3 to 17.2%) in patients with APBA, bronchitis, aspergilloma, or chronic aspergillosis and 4.6% in patients with invasive aspergillosis (5/108; 95% CI, 1.7 to 10.7%). The 6-week survival in invasive aspergillosis was 52.5%, while susceptibility testing revealed azole resistance in only 2/58 of the deceased patients. The clinical impact of Aspergillus fumigatus resistance was limited in our patient population with Aspergillus diseases.

Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library.

The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database.

Reclassification of Staphylococcus jettensis De Bel et al. 2013 as Staphylococcus petrasii subsp. jettensis subsp. nov. and emended description of Staphylococcus petrasii Pantucek et al. 2013.

The type and clinical strains of two recently described coagulase-negative species of the genus Staphylococcus, Staphylococcus petrasii and Staphylococcus jettensis, were compared using dnaJ, tuf, gap, hsp60 and rpoB gene sequences, DNA-DNA hybridization, ribotyping, repetitive sequence-based PCR fingerprinting and extensive biochemical characterization. Based on the results, the species description of S. petrasii has been emended and S. jettensis should be reclassified as a novel subspecies within S. petrasii for which the name Staphylococcus petrasii subsp. jettensis subsp. nov. is proposed. The type strain is SEQ110(T) ( = LMG 26879(T) = CCUG 62657(T) = DSM 26618(T) = CCM 8494(T)).

D-Zone test for detection of inducible clindamycin resistance using SirScan paper disks and Rosco Neo-Sensitabs at 25 and 15 mm distances.

Clindamycin is a possible antibiotic treatment of infections by Gram-positive cocci. However, its use can be limited by inducible clindamycin resistance. To screen for the presence of this type of resistance, the D-zone test is used. The aim of this study was to compare the performance of SirScan paper disks with that of Rosco Neo-Sensitabs for the D-zone test at distances according to dispensers provided by the manufacturers (25 mm) and when the disks are placed at a distance of 15 mm. We studied 364 Gram-stain-positive cocci representing clinical isolates that were resistant to erythromycin and susceptible to clindamycin. Out of these isolates, 207 (57%) gave a positive D-zone test result at 25 mm distance using SirScan paper disks. When the test was repeated with the same disks placed 15 mm from the 157 (43%) isolates that had previously given a negative result, 58 (36.9%) gave a positive D-zone test result. The same isolates were also found to test positive when Rosco Neo-Sensitabs were used. Placing the disks at a distance of 15 mm instead of 25 mm led to an 84.3% increase in positive D-tests among Staphylococcus aureus, 43.8% among group B streptococci (GBS) and 6.4% among coagulase-negative staphylococci (CNS). In conclusion, the SirScan paper disks are equivalent to Rosco Neo-Sensitabs in screening for inducible resistance to clindamycin. The D-test needs to be performed at a shorter distance (15 mm) to prevent false-negative reporting.

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity.

BACKGROUND: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. RESULTS: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1).To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. CONCLUSIONS: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Sampling and decontamination method for culture of nontuberculous mycobacteria in respiratory samples of cystic fibrosis patients.

We confirmed that chlorhexidine decontamination yielded more nontuberculous mycobacteria than did the N-acetyl-l-cysteine-NaOH-oxalic acid procedure from respiratory samples of cystic fibrosis patients on solid cultures. However, this improved recovery is mostly balanced if the latter is combined with liquid culture. Furthermore, none of the 145 cough swabs, used to sample young children, cultured positive, suggesting that swabs are low-quality samples.

RegaDB: community-driven data management and analysis for infectious diseases.

SUMMARY: RegaDB is a free and open source data management and analysis environment for infectious diseases. RegaDB allows clinicians to store, manage and analyse patient data, including viral genetic sequences. Moreover, RegaDB provides researchers with a mechanism to collect data in a uniform format and offers them a canvas to make newly developed bioinformatics tools available to clinicians and virologists through a user friendly interface. AVAILABILITY AND IMPLEMENTATION: Source code, binaries and documentation are available on http://rega.kuleuven.be/cev/regadb. RegaDB is written in the Java programming language, using a web-service-oriented architecture.

Staphylococcus jettensis sp. nov., a coagulase-negative staphylococcal species isolated from human clinical specimens.

Eight coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human clinical specimens at two different Belgian medical facilities. All strains were non-motile, Gram-stain-positive, catalase-positive cocci. DNA G+C content, peptidoglycan type, menaquinone pattern, the presence of teichoic acid and cellular fatty acid composition were in agreement with the characteristics of species of the genus Staphylococcus. Sequencing of the 16S rRNA gene and four housekeeping genes (dnaJ, tuf, gap and rpoB) demonstrated that these strains constitute a separate taxon within the genus Staphylococcus. Less than 41% DNA-DNA hybridization with the most closely related species of the genus Staphylococcus (Staphylococcus haemolyticus, Staphylococcus hominis and Staphlococcus lugdunensis) was observed. Key biochemical characteristics that allowed these bacteria to be distinguished from their nearest phylogenetic neighbours are arginine dihydrolase positivity, ornithine decarboxylase negativity and inability to produce acid aerobically from D-mannose, α-lactose and turanose. Acid is produced aerobically from trehalose. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus jettensis sp. nov. The type strain is SEQ110(T) ( =LMG 26879(T) =CCUG 62657(T) =DSM 26618(T)).

HIV-1 low-level viraemia assessed with 3 commercial real-time PCR assays show high variability.

BACKGROUND: Current real-time PCR-based HIV-1 viral load (VL) assays allow the detection of residual viraemia in antiretroviral-treated patients. The clinical outcome of HIV1 patients experiencing low-level replication (<50 cop/mL) in comparison with fully suppressed patients is currently debated. We analysed variability of 3 VL assays <50 cop/mL, and evaluated the reproducibility of viral blips <100 cop/mL. METHODS: Three commercial VL assays were tested: Versant HIV-1 RNA 1.0 kPCR (Siemens), Abbott Realtime HIV-1, and Cobas Ampliprep/Cobas Taqman HIV-1 v2.0 (Roche). Ten replicates of a reference sample at 4 low target dilutions were tested to evaluate assay variability. Prospective collection of 181 clinical samples with detectable VL <50 cop/mL was used to evaluate intra-and inter-assay variability by triplicate testing. Samples from 26 patients experiencing a viral blip were retested. RESULTS: All assays showed substantial variability at low VL level: the coefficient of variation at 100, 50, 25 and 12 cop/mL ranged respectively from 32 to 44%, 35 to 68%, 41 to 83% and 33 to 77%. In the intra-assay evaluation of repeatability, 52.5 to 57.5% of detectable VL <50 cop/mL tested in triplicate showed at least one fully undetected result. Variability was similar in the inter-assay arm. The VL blips could only be reproduced in 19% of cases. CONCLUSIONS: The most recent versions of widespread commercial VL assays showed substantial variability at low levels and residual viraemia could not be consistently reproduced. Patient outcome studies comparing residual VL to full suppression are therefore biased when using commercial assays.

Prosthetic valve endocarditis caused by Bordetella holmesii, an Acinetobacter lookalike.

We report a case of fulminant endocarditis on a prosthetic homograft aortic valve caused by Bordetella holmesii, which was successfully managed by surgical valve replacement and antibiotic treatment. B. holmesii, a strictly aerobic, small, Gram-negative coccobacillus, has been implicated as an infrequent cause of a pertussis-like syndrome and other respiratory illnesses. However, B. holmesii is also a rare cause of septicaemia and infective endocarditis, mostly in immunocompromised patients. To our knowledge, this is the first report of B. holmesii endocarditis on a prosthetic aortic valve. Routine laboratory testing initially misidentified the strain as Acinetobacter sp. Correct identification was achieved by 16S rRNA gene and outer-membrane protein A (ompA) gene sequencing. Interestingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry also produced an accurate species-level identification. Subsequent susceptibility testing and review of the literature revealed ceftazidime, cefepime, carbapenems, aminoglycosides, fluoroquinolones, piperacillin/tazobactam, tigecycline and colistin as possible candidates to treat infections caused by B. holmesii.

Species identification of clinical Prevotella isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The performance of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database.

Antimicrobial susceptibility of Streptococcus pneumoniae isolates from vaccinated and non-vaccinated patients with a clinically confirmed diagnosis of community-acquired pneumonia in Belgium.

We assessed the in vitro susceptibility of Streptococcus pneumoniae isolates from patients with confirmed community-acquired pneumonia (CAP) to ß-lactams, macrolides and fluoroquinolones and the association of non-susceptibility and resistance with serotypes/serogroups (STs/SGs), patient's risk factors and vaccination status. Samples (blood or lower respiratory tract) were obtained in 2007-2009 from 249 patients (from seven hospitals in Belgium) with a clinical and radiological diagnosis of CAP [median age 61 years (11.6% aged <5 years); 85% without previous antibiotic therapy; 86% adults with level II Niederman's severity score]. MIC determination (EUCAST breakpoints) showed for: (i) amoxicillin, 6% non-susceptible; cefuroxime (oral), 6.8% resistant; (ii) macrolides: 24.9% erythromycin-resistant [93.5% erm(B)-positive] but 98.4% telithromycin-susceptible; and (iii) levofloxacin and moxifloxacin, all susceptible. Amongst SGs: ST14, all resistant to macrolides and most intermediate to ß-lactams; SG19 (>94% ST19A), 73.5% resistant to macrolides and 18-21% intermediate to ß-lactams; and SG6, 33% resistant to clarithromycin. Apparent vaccine failures: 3/17 for 7-valent vaccine (children; ST6B, 23F); 16/29 for 23-valent vaccine (adults ST3, 7F, 12F, 14, 19A, 22F, 23F, 33F). Isolates from nursing home residents, hospitalised patients and patients with non-respiratory co-morbidities showed increased MICs for amoxicillin, all ß-lactams, and ß-lactams and macrolides, respectively. Regarding antibiotic susceptibilities: (i) amoxicillin is still useful for empirical therapy but with a high daily dose; (ii) cefuroxime axetil and macrolides (but not telithromycin) are inappropriate for empirical therapy; and (iii) moxifloxacin and levofloxacin are the next 'best empirical choice' (no resistant isolates) but levofloxacin will require 500 mg twice-daily dosing for effective coverage.

A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption.

In a phase I/IIa clinical trial, 17 HIV-1 infected patients, stable on cART, received 4 vaccinations with autologous dendritic cells electroporated with mRNA encoding Tat, Rev and Nef, after which cART was interrupted. Vaccination was safe and feasible. During the analytical treatment interruption (ATI), no serious adverse events were observed. Ninety-six weeks following ATI, 6/17 patients remained off therapy. Although induced and/or enhanced CD4(+) and CD8(+) T-cell responses specific for the immunogens were observed in most of the patients, we found no correlation with the number of weeks off cART. Moreover, CD4(+) T-cell counts, plasma viral load and the time remaining off cART following ATI did not differ from historical control data. To conclude, the vaccine was safe, well tolerated and resulted in vaccine-specific immune responses. Since no correlation with clinical parameters could be found, these results warrant further research in order to optimize the efficacy of vaccine-induced T-cell responses.

Case report: Infrapatellar bursitis caused by Prototheca wickerhamii.

A 54-year-old immunocompetent man presented with an infrapatellar bursitis caused by Prototheca wickerhamii. Because of clinical and microbiological relapse two weeks after bursectomy, six weekly injections of 5 mg of conventional amphotericin B were chosen for intrabursal treatment. Four months after completion of the treatment, the patient remains cured.

Differentiation of cfiA-negative and cfiA-positive Bacteroides fragilis isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Carbapenem resistance in Bacteroides fragilis is associated with cfiA-encoded class B metallo-beta-lactamase. cfiA-negative and cfiA-positive isolates belong to genotypically distinct groups. Of a total of 248 B. fragilis isolates included in this study, 214 were susceptible, 10 were intermediate, and 24 were resistant to meropenem. We show that matrix-assisted laser desorption ionization-time of flight mass spectrometry is able to differentiate between cfiA-negative and cfiA-positive isolates and predict carbapenem resistance in a routine laboratory setting.