Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

BACKGROUND: Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. METHODS: In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. RESULTS: Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). CONCLUSIONS: Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

Enhancement and Comparison of (Ceftazidime-)Avibactam Plus Aztreonam Susceptibility Tests for Stenotrophomonas maltophilia in Clinical Diagnostics.

Stenotrophomonas maltophilia is naturally resistant to many antimicrobials. We evaluated the in vitro activity and reproducibility of two different super-position methods of aztreonam in combination with ceftazidime-avibactam for S. maltophilia and compared these results with the recently available aztreonam-avibactam gradient strip. We recommend an improved super-position method that avoids the possible risk of handling a contaminated aztreonam strip. In addition, we report that the cefazidime-avibactam and aztreonam super-position method showed increased in vitro activity in comparison with aztreonam-avibactam indicating activity of the ceftazidime component in vitro.

A prospective multicentre screening study on multidrug-resistant organisms in intensive care units in the Dutch-German cross-border region, 2017 to 2018: the importance of healthcare structures.

BackgroundAntimicrobial resistance poses a risk for healthcare, both in the community and hospitals. The spread of multidrug-resistant organisms (MDROs) occurs mostly on a local and regional level, following movement of patients, but also occurs across national borders.AimThe aim of this observational study was to determine the prevalence of MDROs in a European cross-border region to understand differences and improve infection prevention based on real-time routine data and workflows.MethodsBetween September 2017 and June 2018, 23 hospitals in the Dutch (NL)-German (DE) cross-border region (BR) participated in the study. During 8 consecutive weeks, patients were screened upon admission to intensive care units (ICUs) for nasal carriage of meticillin-resistant Staphylococcus aureus (MRSA) and rectal carriage of vancomycin-resistant Enterococcus faecium/E. faecalis (VRE), third-generation cephalosporin-resistant Enterobacteriaceae (3GCRE) and carbapenem-resistant Enterobacteriaceae (CRE). All samples were processed in the associated laboratories.ResultsA total of 3,365 patients were screened (median age: 68 years (IQR: 57-77); male/female ratio: 59.7/40.3; NL-BR: n = 1,202; DE-BR: n = 2,163). Median screening compliance was 60.4% (NL-BR: 56.9%; DE-BR: 62.9%). MDRO prevalence was higher in DE-BR than in NL-BR, namely 1.7% vs 0.6% for MRSA (p = 0.006), 2.7% vs 0.1% for VRE (p < 0.001) and 6.6% vs 3.6% for 3GCRE (p < 0.001), whereas CRE prevalence was comparable (0.2% in DE-BR vs 0.0% in NL-BR ICUs).ConclusionsThis first prospective multicentre screening study in a European cross-border region shows high heterogenicity in MDRO carriage prevalence in NL-BR and DE-BR ICUs. This indicates that the prevalence is probably influenced by the different healthcare structures.

Should all hip and knee prosthetic joints be aspirated prior to revision surgery?

AIMS: It is essential to exclude a periprosthetic joint infection (PJI) prior to revision surgery. It is recommended to routinely aspirate the joint before surgery. However, this may not be necessary in a subgroup of patients. The aim of our study was to investigate if specific clinical and implant characteristics could be identified to rule out a PJI prior to revision surgery. METHODS: We retrospectively evaluated clinical and implant characteristics of patients who underwent a hip or knee revision surgery between October 2015 and October 2018. Patients were diagnosed with a PJI according to the MSIS diagnostic criteria. RESULTS: A total of 156 patients were analyzed, including 107 implants that were revised because of prosthetic loosening and 49 because of mechanical failure (i.e. instability, malalignment or malpositioning). No PJI was diagnosed in the group with mechanical failure. In the prosthetic loosening group, 20 of 107 were diagnosed with a PJI (19%). Although there was a significantly lower chance of having a PJI with an implant age of > 5 years combined with a CRP < 5 mg/L, an infection was still present in 3 out of 39 cases (8%). CONCLUSION: Implants with solely mechanical failure without signs of loosening and low inflammatory parameters probably do not require a synovial fluid aspiration. These results need to be confirmed in a larger cohort of patients. In case of prosthetic loosening, all joints need to be aspirated before surgery as no specific characteristic could be identified to rule out an infection.

Paradoxal Trends in Azole-Resistant Aspergillus fumigatus in a National Multicenter Surveillance Program, the Netherlands, 2013-2018.

We investigated the prevalence of azole resistance of Aspergillus fumigatus isolates in the Netherlands by screening clinical A. fumigatus isolates for azole resistance during 2013-2018. We analyzed azole-resistant isolates phenotypically by in vitro susceptibility testing and for the presence of resistance mutations in the Cyp51A gene. Over the 6-year period, 508 (11%) of 4,496 culture-positive patients harbored an azole-resistant isolate. Resistance frequency increased from 7.6% (95% CI 5.9%-9.8%) in 2013 (58/760 patients) to 14.7% (95% CI 12.3%-17.4%) in 2018 (112/764 patients) (p = 0.0001). TR34/L98H (69%) and TR46/Y121F/T289A (17%) accounted for 86% of Cyp51A mutations. However, the mean voriconazole MIC of TR34/L98H isolates decreased from 8 mg/L (2013) to 2 mg/L (2018), and the voriconazole-resistance frequency was 34% lower in 2018 than in 2013 (p = 0.0001). Our survey showed changing azole phenotypes in TR34/L98H isolates, which hampers the use of current PCR-based resistance tests.

A Second Surgical Debridement for Acute Periprosthetic Joint Infections Should Not Be Discarded.

BACKGROUND: In acute periprosthetic joint infections (PJIs), a second surgical debridement (debridement, antibiotics, and implant retention [DAIR]) is generally not recommended after a failed first one. We identified the failure rate of a second DAIR and aimed to identify patients in whom an additional debridement might still be beneficial. METHODS: Patients with acute PJI of the hip or knee and treated with DAIR between 2006 and 2016 were retrospectively evaluated. A second DAIR was routinely performed provided that the soft tissue was intact. Failure of a second DAIR was described as (1) the need for additional surgical intervention to achieve infection control, (2) the need for antibiotic suppressive therapy due to persistent clinical and/or biochemical signs of infection, or (3) PJI related death. RESULTS: From the 455 cases treated with DAIR, 144 cases underwent a second debridement (34.6%). Thirty-seven cases failed (37/144, 25.7%). The implant needed to be removed in 23 cases (23/144, 16%). Positive cultures during the second DAIR (odds ratio 3.16, 95% confidence interval 1.29-7.74) and chronic renal insufficiency (odds ratio 13.6, 95% confidence interval 2.03-91.33) were independent predictors for failure in the multivariate analysis. No difference in failure was observed between persistent infection with the same microorganism and reinfection with a new microorganism (failure rate 31.6% vs 34.6%, P = .83). CONCLUSION: A second DAIR had a low failure rate in our cohort of patients and the implant could be retained in the majority of them. Therefore, a second DAIR should not be discarded in acute PJIs.

Use of gentamicin-impregnated beads or sponges in the treatment of early acute periprosthetic joint infection: a propensity score analysis.

Objectives: Early acute periprosthetic joint infections (PJIs) treated with debridement, antibiotics and implant retention (DAIR) have failure rates ranging from 10% to 60%. We determined the efficacy of applying local gentamicin-impregnated beads and/or sponges during debridement in early PJI. Methods: Patients with early acute PJI, defined as less than 21 days of symptoms and treated with DAIR within 90 days after index surgery, were retrospectively evaluated. Early failure was defined as PJI-related death, the need for implant removal or a second DAIR or antibiotic suppressive therapy owing to persistent signs of infection, all within 60 days after initial debridement. Overall failure was defined as implant removal at any timepoint during follow-up. A 1:1 propensity score matching was performed to correct for confounding factors. Results: A total of 386 patients were included. Local gentamicin was applied in 293 patients (75.9%) and was withheld in 93 patients (24.1%). Multivariate analysis demonstrated that the use of local gentamicin was independently associated with early failure (OR = 1.97, 95% CI = 1.12-3.48). After propensity matching, early failure was 40.3% in the gentamicin group versus 26.0% in the control group (P = 0.06) and overall failure was 5.2% in the gentamicin group versus 2.6% in the control group (P = 0.40). These numbers remained when solely analysing the application of gentamicin-impregnated sponges. Conclusions: Even after propensity score matching, failure rates remained higher if local gentamicin-impregnated beads and/or sponges were administered in early acute PJI. Based on these results, their use should be discouraged.

Susceptibility of ESBL Escherichia coli and Klebsiella pneumoniae to fosfomycin in the Netherlands and comparison of several testing methods including Etest, MIC test strip, Vitek2, Phoenix and disc diffusion.

Objectives: Fosfomycin susceptibility testing is complicated and prone to error. Before using fosfomycin widely in patients with serious infections, acquisition of WT distribution data and reliable susceptibility testing methods are crucial. In this study, the performance of five methods for fosfomycin testing in the routine laboratory against the reference method was evaluated. Methods: Ten laboratories collected up to 100 ESBL-producing isolates each (80 Escherichia coli and 20 Klebsiella pneumoniae). Isolates were tested using Etest, MIC test strip (MTS), Vitek2, Phoenix and disc diffusion. Agar dilution was performed as the reference method in a central laboratory. Epidemiological cut-off values (ECOFFs) were determined for each species and susceptibility and error rates were calculated. Results: In total, 775 E. coli and 201 K. pneumoniae isolates were tested by agar dilution. The ECOFF was 2 mg/L for E. coli and 64 mg/L for K. pneumoniae. Susceptibility rates based on the EUCAST breakpoint of ≤32 mg/L were 95.9% for E. coli and 87.6% for K. pneumoniae. Despite high categorical agreement rates for all methods, notably in E. coli, none of the alternative antimicrobial susceptibility testing methods performed satisfactorily. Due to poor detection of resistant isolates, very high error rates of 23.3% (Etest), 18.5% (MTS), 18.8% (Vitek2), 12.5% (Phoenix) and 12.9% (disc diffusion) for E. coli and 22.7% (Etest and MTS), 16.0% (Vitek2) and 12% (Phoenix) for K. pneumoniae were found. None of the methods adequately differentiated between WT and non-WT populations. Conclusions: Overall, it was concluded that none of the test methods is suitable as an alternative to agar dilution in the routine laboratory.

The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance. A nationwide survey and rationale for the DB-MSG 002 study protocol.

Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres.

Puncture Protocol in the Diagnostic Work-Up of a Suspected Chronic Prosthetic Joint Infection of the Hip.

BACKGROUND: Diagnosing a chronic prosthetic joint infection (PJI) can be challenging. We hypothesized that obtaining preoperative tissue samples for culture in hip arthroplasty will increase the likelihood of diagnosing an infection before revision surgery. The aim of this cohort study was to determine the diagnostic accuracy of 2 tissue acquiring biopsy strategies to diagnose a PJI. METHODS: Patients with a painful hip arthroplasty, in which a chronic PJI was suspected, were included. Tissue samples were obtained either by ultrasound guidance with a 16-Gauge needle (2012-2013) or in the operating room with a thick-bore needle (2013-2016). Revision surgery tissue biopsies were used as the gold standard. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were calculated. RESULTS: A total of 16 patients in the ultrasound cohort and 29 patients in the surgical cohort were included. Thirty-one percent (n = 14) were finally diagnosed with a PJI. The addition of thick bore needle tissue biopsies resulted in 9% more diagnosed PJIs compared with synovial fluid alone. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio was 33%, 85%, 33%, 85%, 2.2, and 0.8, respectively, for the ultrasound-guided biopsy cohort and 82%, 100%, 100%, 90%, infinite, and 0.2, respectively, for the surgical biopsy cohort. CONCLUSION: Obtaining multiple good quality tissue biopsies in a sterile environment will contribute to the diagnosis of a chronic PJI of the hip, with a higher diagnostic accuracy compared with ultrasound-guided thin needle biopsies and compared with synovial fluid culture alone.

Synovial Calprotectin: An Inexpensive Biomarker to Exclude a Chronic Prosthetic Joint Infection.

BACKGROUND: To diagnose or exclude a chronic prosthetic joint infection (PJI) can be a clinical challenge. Therefore, sensitive and specific biomarkers are needed in the diagnostic work-up. Calprotectin is a protein with antimicrobial properties and is released by activated neutrophils, making it a specific marker for infection. Because of its low costs and ability to obtain a quantitative value as a point of care test, it is an attractive marker to use in clinical practice. In addition, the test is already used in routine care in most hospitals for other indications and therefore easy to implement. METHODS: Between June 2015 and June 2017 we collected synovial fluid of all consecutive patients who underwent revision surgery of a prosthetic joint because of chronic pain with or without prosthetic loosening. Synovial calprotectin was measured using a lateral flow immunoassay. A PJI was defined by the diagnostic criteria described by the Musculoskeletal Infection Society. RESULTS: Fifty-two patients with chronic pain were included. A PJI was diagnosed in 15 of 52 (29%) patients. The median calprotectin in the PJI group was 859 mg/L (interquartile range 86-1707) vs 7 mg/L (interquartile range 3-25) in the control group (P < .001). With a cut-off value of 50 mg/L, synovial calprotectin showed a sensitivity, specificity, positive predictive value, and negative predictive value of 86.7%, 91.7%, 81.3%, and 94.4%, respectively. CONCLUSION: Synovial calprotectin is a useful and cheap biomarker to use in the diagnostic work-up of patients with chronic pain, especially to exclude a PJI prior to revision surgery.

18F-FDG-PET uptake in non-infected total hip prostheses.

Background and purpose - 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) can be used in the diagnostic work-up of a patient with suspected periprosthetic joint infection (PJI) but, due to a lack of accurate interpretation criteria, this technique is not routinely applied. Since the physiological uptake pattern of FDG around a joint prosthesis is not fully elucidated, we determined the physiological FDG uptake in non-infected total hip prostheses. Patients and methods - Patients treated with primary total hip arthroplasty (1995-2016) who underwent a FDG-PET/CT for an indication other than a suspected PJI were retrospectively evaluated. Scans were both visually and quantitatively analyzed. Semi-quantitative analysis was performed by calculating maximum and peak standardized uptake values (SUVmax and SUVpeak) by volume of interests (VOIs) at 8 different locations around the prosthesis. Results - 58 scans from 30 patients were analyzed. In most hips, a diffuse heterogeneous uptake pattern around the prosthesis was observed (in 32/38 of the cemented prostheses, and in 16/20 of the uncemented prostheses) and most uptake was located around the neck of the prosthesis. The median SUVmax in the cemented group was 2.66 (95% CI 2.51-3.10) and in the uncemented group 2.87 (CI 2.65-4.63) (Median difference = -0.36 [CI -1.2 to 0.34]). In uncemented prostheses, there was a positive correlation in time between the age of the prosthesis and the FDG uptake (rs = 0.63 [CI 0.26-0.84]). Interpretation - Our study provides key data to develop accurate interpretation criteria to differentiate between physiological uptake and infection in patients with a prosthetic joint.

Emerging pan-resistance in Trichosporon species: a case report.

BACKGROUND: Trichosporon species are ubiquitously spread and known to be part of the normal human flora of the skin and gastrointestinal tract. Trichosporon spp. normally cause superficial infections. However, in the past decade Trichosporon spp. are emerging as opportunistic agents of invasive fungal infections, particularly in severely immunocompromised patients. Clinical isolates are usually sensitive to triazoles, but strains resistant to multiple triazoles have been reported. CASE PRESENTATION: We report a high-level pan-azole resistant Trichosporon dermatis isolate causing an invasive cholangitis in a patient after liver re-transplantation. This infection occurred despite of fluconazole and low dose amphotericin B prophylaxis, and treatment with combined liposomal amphotericin B and voriconazole failed. CONCLUSION: This case and recent reports in literature show that not only bacteria are evolving towards pan-resistance, but also pathogenic yeasts. Prudent use of antifungals is important to withstand emerging antifungal resistance.

Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles.

BACKGROUND: Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus. METHODS: Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles. RESULTS: Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR46/Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR46/Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR46/Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR46/Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites. CONCLUSIONS: We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasive aspergillosis. Recovery of TR46/Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.

A high prevalence of Cutibacterium acnes infections in scoliosis revision surgery, a diagnostic and therapeutic dilemma.

PURPOSE: To investigate if serum inflammatory markers or nuclear imaging can accurately diagnose a chronic spinal instrumentation infection (SII) prior to surgery. METHODS: All patients who underwent revision of spinal instrumentation after a scoliosis correction between 2017 and 2019, were retrospectively evaluated. The diagnostic accuracy of serum C-reactive protein (CRP) and Erythrocyte Sedimentation Rate (ESR), 18F-fluorodeoxyglucose positron emission tomography combined with computed tomography (FDG-PET/CT) and Technetium-99m-methylene diphosphonate (99mTc-MDP) 3-phase bone scintigraphy (TPBS) to diagnose infection were studied. Patients with an acute infection or inadequate culture sampling were excluded. SII was diagnosed if ≥ 2 of the same microorganism(s) were isolated from intra-operative tissue cultures. RESULTS: 30 patients were included. The indication for revision surgery was pseudoarthrosis in the majority of patients (n = 15). 22 patients (73%) were diagnosed with SII. In all infected cases, Cutibacterium acnes was isolated, including 5 cases with a polymicrobial infection. The majority of patients had low inflammatory parameters preoperatively. For CRP > 10.0 mg/L, the sensitivity was 9.1% and specificity 100%; for ESR > 30 mm/h, the sensitivity was 9.1% and specificity 100%. The diagnostic accuracy for nuclear imaging was 64% for the FDG-PET/CT and 67% for the TPBS to diagnose infection. CONCLUSIONS: The prevalence of SII in patients undergoing revision spinal surgery is high, with Cutibacterium acnes as the main pathogen. No diagnostic tests could be identified that could accurately diagnose or exclude SII prior to surgery. Future studies should aim to find more sensitive diagnostic modalities to detect low-grade inflammation.

First evaluation of a commercial multiplex PCR panel for rapid detection of pathogens associated with acute joint infections.

Background: prompt recognition and identification of the causative microorganism in acute septic arthritis of native and prosthetic joints is vital to increase the chances of successful treatment. The aim of this study was to independently assess the diagnostic accuracy of the multiplex BIOFIRE® Joint Infection (JI) Panel (investigational use only) in synovial fluid for rapid diagnosis. Methods: synovial fluid samples were collected at the University Medical Center Groningen from patients who had a clinical suspicion of a native septic arthritis, early acute (post-operative, within 3 months after arthroplasty) periprosthetic joint infection (PJI) or late acute (hematogenous, ≥ 3 months after arthroplasty) PJI. JI Panel results were compared to infection according to Musculoskeletal Infection Society criteria and culture-based methods as reference standard. Results: a total of 45 samples were analysed. The BIOFIRE JI Panel showed a high specificity (100 %, 95 % confidence interval (CI): 78-100) in all patient categories. Sensitivity was 83 % (95 % CI: 44-97) for patients with a clinical suspicion of native septic arthritis ( n = 12 ), 73 % (95 % CI: 48-89) for patients with a clinical suspicion of a late acute PJI ( n = 14 ), and 30 % (95 % CI: 11-60) for patients with a clinical suspicion of an early acute PJI ( n = 19 ). Conclusion: the results of this study indicate a clear clinical benefit of the BIOFIRE JI Panel in patients with a suspected native septic arthritis and late acute (hematogenous) PJI, but a low clinical benefit in patients with an early acute (post-operative) PJI due to the absence of certain relevant microorganisms, such as Staphylococcus epidermidis, from the panel.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus.

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population.

A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 µg/ml, respectively, while amphotericin B (0.24 µg/ml), itraconazole (0.08 µg/ml), voriconazole (0.07 µg/ml), posaconazole (0.06 µg/ml), and isavuconazole (0.03 µg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 µg/ml), while decreased susceptibility (≥16 µg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively.