Changing epidemiology of parvovirus B19 in the Netherlands since 1990, including its re-emergence after the COVID-19 pandemic.

Parvovirus B19V (B19V) infection during pregnancy can be complicated by potentially life-threatening fetal hydrops, which can be managed by intrauterine transfusion (IUT). This study investigates the long-term temporal patterns in the epidemiology of B19V and evaluates the impact on fetal hydrops, by combining data on B19V infections from the Dutch Sentinel Surveillance system in the period 1990 to 2023, Dutch blood banking data and hospital data on fetal hydrops. Using wavelet analysis, we identified annual epidemic cycles in the Netherlands in the period 1990-2019 and we identified superimposed multiannual cycles in the period 1990-2009. After 2009, no multiannual cycle could be identified, although the incidence fluctuated and correlates with number of IUT performed. As of 2020, weekly reports of B19V infection demonstrated a historically low incidence and B19V-DNA positive blood donors were nearly absent. From May 2020 to May 2023, no IUT for B19V-related hydrops was performed. In the spring of 2023, B19V infections re-emerged, reaching pre-pandemic epidemic levels. Due to the changes in B19V epidemiology over the last 30 years and the near-absence of B19V during the COVID-19 pandemic, the resulting low immunity levels may lead to rebound outbreaks. Alertness to severe complications such as fetal hydrops is warranted.

Transient Parvovirus B19 DNAemia After Kidney Transplantation: A 2-Sided Story.

Parvovirus B19 (B19V) DNAemia appears to be a relatively common finding after kidney transplantation. However, not all DNAemia signifies active infection with replicating virus. This study screened 134 patients posttransplantation for B19V DNAemia and identified 2 cases in which viral DNA was present after transplantation, with the donor kidney as probable source of the DNA. In both cases intact viral particles could not be detected using an endonuclease method, indicating the presence of noninfectious DNA remnants.

Highly Divergent SARS-CoV-2 Alpha Variant in Chronically Infected Immunocompromised Person.

We detected a highly divergent SARS-CoV-2 Alpha variant in an immunocompromised person several months after the latest detection of the Alpha variant in the Netherlands. The patient was infected for 42 weeks despite several treatment regimens and disappearance of most clinical symptoms. We identified several potential immune escape mutations in the spike protein.

Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

INTRODUCTION: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. METHODS: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. RESULTS: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. CONCLUSIONS: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

Evaluation of the Atellica® UAS 800: a new member of the automated urine sediment analyzer family.

BACKGROUND: In 2017 the Atellica® UAS 800 urine sediment analyzer was introduced by Siemens Healthineers. We investigated its applicability in the standardization and automation of the laboratory urinalysis workflow, including the prediction of urine culture outcome and glomerular pathology. METHODS: We evaluated the performance characteristics of the Atellica® UAS 800 and its correlation with the iQ200 (Beckman Coulter). In addition, we studied the agreement between Atellica® UAS 800 and CLINITEK Novus® and determined the predictive value of bacteria and leukocyte counts for urine culture outcome. Furthermore, we investigated the ability of Atellica® UAS 800 to identify pathological casts and dysmorphic erythrocytes in comparison to manual microscopy. RESULTS: Erythrocyte and leukocyte analyses indicated high intra- and inter-run precisions and good correlations with the iQ200. We found that the Atellica® UAS 800 detects bacteria with higher sensitivity than the iQ200. The Atellica® UAS 800 and CLINITEK Novus® showed a high degree of conformity. We determined seven combinations of clinical cut-off values of bacteria and leukocytes for predicting urine culture outcome with sensitivity, specificity, and negative predictive values of 95%, 52%, and 93%, respectively. Using the Atellica® UAS 800, hyaline casts, erythrocyte casts, leukocyte casts, and dysmorphic erythrocytes were correctly recognized in 76%, 22%, 2%, and 39% of the samples, respectively. CONCLUSIONS: The Atellica® UAS 800 is a robust, fast, and user-friendly analyzer, which accurately quantifies erythrocytes, leukocytes, bacteria and squamous epithelial cells, and may be utilized for predicting positive urine cultures. The detection of clinically important pathological casts and dysmorphic erythrocytes proved insufficient.

Parvovirus B19 DNA detectable in hearts of patients with dilated cardiomyopathy, but absent or inactive in blood.

AIMS: Parvovirus B19 (B19V) is often assumed to be a cause of dilated cardiomyopathy (DCM), based on the quantification of B19V DNA in endomyocardial biopsies (EMB). Whether the presence of B19V DNA correlates with active infection is still debated. Application of the enzyme endonuclease to blood samples results in degradation of B19V DNA remnants but leaves viral particles intact, which enables differentiation between active and past infection. In this study, the susceptibility to degradation by endonuclease of B19V DNA in blood was compared between DCM patients and a control group of recent B19V infections. METHODS AND RESULTS: Twenty blood samples from 20 adult patients with DCM, who previously tested positive for B19V DNA in EMB and/or blood, were tested with B19V PCR before and after application of endonuclease to the samples. Six blood samples tested positive for B19V DNA with a mean viral load of 2.3 × 104  IU/mL. In five samples, B19V DNA became undetectable after endonuclease (100% load reduction); in one sample DNA load showed a 23% log load reduction (viral load before endonuclease: 9.1 × 104  IU/mL; after: 6.5 × 103  IU/mL). Presence of cardiac inflammation did not differ between patients with B19V DNAemia (1/4) and patients without B19V DNAemia (6/14) (P value = 1.0). In all 18 control samples of proven recent B19V infections, DNA remained detectable after application of endonuclease, showing only a mean log load reduction of 2.3% (mean viral load before endonuclease: 8.1 × 1011  IU/mL; after: 8.0 × 1011  IU/mL). Load reduction differed significantly between the DCM group and the control group; indicating the presence of intact viral particles in the control group with proven active infection and the presence of DNA remnants in the DCM group (P value = 0.000). CONCLUSION: During recent B19V infection, viral DNA levels in blood were unaffected by endonuclease. In contrast, B19V DNA in blood in patients with DCM became undetectable or strongly reduced after application of endonuclease. Circulating viral DNA in this subset of patients with presumed parvovirus-associated DCM does not consist of intact viral particles. Viral replicative activity cannot be assumed from demonstrating B19V DNA in cardiac tissue or in blood in DCM patients.

Diagnosis of intrauterine parvovirus B19 infection at birth - Value of DNA detection in neonatal blood and dried blood spots.

BACKGROUND: Diagnosis of congenital viral infection at birth is generally attempted by direct detection of the virus by PCR in various neonatal materials. How to reliably diagnose intrauterine infection with parvovirus B19 (B19 V) at birth is unknown. OBJECTIVES: To evaluate the performance of B19 V DNA detection in cord blood (CB) or neonatal dried blood spots (DBS) in diagnosing fetal infection. STUDY DESIGN: Two cohorts of children diagnosed prenatally with an intrauterine B19 V infection were included in this study. CB samples of intrauterine B19 V infections that were sent to a reference laboratory for congenital infections in Stuttgart, Germany in the period 1995-2014 were tested in triplicate for B19 V DNA by quantitative PCR. DBS from children with intrauterine B19 V infection that underwent IUT at the LUMC, Leiden, the Netherlands in the period 2009-2014 were tested for B19 V DNA by quantitative B19 V PCR in triplicate. RESULTS: Fourteen of twenty (70 %) CB samples tested positive for B19 V DNA. The positivity rate was 40 % (4/10) in those with a prenatal diagnosis <20 weeks gestation. When intrauterine B19 V infection was diagnosed thereafter, 100 % (10/10) samples were B19 V DNA positive. Of the thirteen available DBS, twelve (92 %) tested positive. Viral load in CB and DBS corresponded inversely with time from fetal diagnosis to birth. CONCLUSION: B19 V DNA can be detected in neonatal blood samples of children following intrauterine B19 V infection, although the possibility of false-negatives, even in severe infections, should be considered. B19 V viral load at birth correlates with timing of infection.

Use of Automated Urine Microscopy Analysis in Clinical Diagnosis of Urinary Tract Infection: Defining an Optimal Diagnostic Score in an Academic Medical Center Population.

A retrospective case record study was conducted that established a scoring tool based on clinical and iQ200 parameters, able to predict or rule out the clinical diagnosis of UTI in the majority of adult patients in an academic hospital. Automated standardized quantitative urine analysis, such as iQ200 analysis, is on the rise because of its high accuracy and efficiency compared to those of traditional urine analysis. Previous research on automated urinalysis focused mainly on predicting culture results but not on the clinical diagnosis of urinary tract infection (UTI). A retrospective analysis was conducted of consecutive urine samples sent in for culture because of suspected UTI. UTI was defined by expert opinion, based on reported symptoms, conventional urine sediment analysis, and urine cultures. Parameters of iQ200 analysis and clinical symptoms and signs were compared between cases and controls. Optimal cutoff values were determined for iQ200 parameters, and multivariate logistic regression analysis was used to identify the set of variables that best predicts the clinical diagnosis of UTI for development of a scoring tool. A total of 382 patients were included. Optimal cutoff values of iQ200 analysis were 74 white blood cells (WBC)/µl, 6,250 "all small particles" (ASP)/µl, and a bacterial score of 2 on an ordinal scale of 0 to 5. The scoring tool attributed 1 point for frequent micturition or increased urge, 2 points for dysuria, 1 point for a bacterial score of ≥2, 2 points for WBC/µl of ≥50, and an additional point for WBC/µl of ≥150. This score had a sensitivity of 86% and a specificity of 92% when using a threshold of <4 points. The combination of iQ200 analysis and a simple survey could predict or rule out UTIs in a majority of patients in an academic medical center.

Necrotising fasciitis as atypical presentation of infection with emerging Neisseria meningitidis serogroup W (MenW) clonal complex 11, the Netherlands, March 2017.

In March 2017, a patient with necrotising fasciitis caused by Neisseria meningitidis serogroup W (MenW) clonal complex 11 was diagnosed in the Netherlands. Unusual and severe presentations of MenW infections are common in the current European epidemic. In the Netherlands, the incidence of MenW infections increased 10-fold, from an average of 0.03 per 100,000 population in 2002-2014 to 0.29 in 2016. Awareness of atypical presentations enables timely adequate treatment and public health action.

Emerging aspergillosis by azole-resistant Aspergillus fumigatus at an intensive care unit in the Netherlands, 2010 to 2013.

The prevalence of invasive aspergillosis (IA) at the intensive care unit (ICU) is unknown and difficult to assess since IA also develops in patients lacking specific host factors. In the Netherlands, increasing azole-resistance in Aspergillus fumigatus complicates treatment of patients with IA. The aim of this study was to determine the prevalence of IA by azole-resistant A. fumigatus at the ICU among patients receiving antifungal treatment and to follow their clinical outcome and prognosis. A retrospective cohort study was conducted in a university hospital ICU from January 2010 to December 2013. From all patients who received antifungal treatment for suspected IA, relevant clinical and microbiological data were collected using a standardised questionnaire. Of 9,121 admitted ICU-patients, 136 had received antifungal treatment for suspected IA, of which 38 had a positive A. fumigatus culture. Ten of the 38 patients harboured at least one azole-resistant isolate. Resistance mechanisms consisted of alterations in the cyp51A gene, more specific TR34/L98H and TR46/T289A/Y121F. Microsatellite typing did not show clonal relatedness, though isolates from two patients were genetically related. The overall 90-day mortality of patients with IA by azole-resistant A. fumigatus and patients with suspicion of IA by azole-susceptible isolates in the ICU was 100% (10/10) vs 82% (23/28) respectively. We conclude that the changing pattern of IA in ICU patients requires appropriate criteria for recognition, diagnosis and rapid resistance tests. The increase in azole resistance rates also challenges a reconsideration of empirical antifungal therapy.

[Legionella pneumonia after the use of CPAP equipment]. / Legionella-pneumonie na gebruik van CPAP-apparatuur.

Continuous positive airway pressure (CPAP) equipment can be colonised by Legionellae and might cause Legionella pneumonia in the user. However, there is no reported case of Legionella pneumonia related to CPAP equipment in which an identical Legionella was found in both the patient and the CPAP equipment. A 51-year-old man came to the Emergency Department with fever, confusion and dyspnoea that had been present for 3 days. His medical history included obstructive sleep apnoea, for which he had been using CPAP therapy at home for 10 weeks. The CPAP equipment showed signs of poor maintenance. Chest X-ray revealed a pulmonary consolidation. Laboratory investigation resulted in a positive urine antigen test for Legionella. Water from the CPAP equipment and sputum from the patient revealed Legionella pneumophila. Serotyping and sequence-based typing showed an identical L. pneumophila serotype 1 ST37. It is important to be aware that CPAP equipment can be colonised with Legionellae and might cause Legionella pneumonia. It is therefore necessary to ask about CPAP therapy in a patient with community-acquired pneumonia.

Interlaboratory Collaboration for Optimized Screening for Urinary Tract Infection.

As the majority of urine samples submitted for culture yields a negative result, rapid screening that accurately predicts culture outcome benefits clinicians by reducing the time to result and improves the efficiency of the microbiological laboratory. Automated urinalysis using the IRIS Diagnostics iQ200 Elite (iQ200) analyzer permits just such a fast and large-scale screening. We aimed to predict and thus to reduce negative cultures with a screening algorithm based on iQ200 urinalysis in a tertiary university hospital. In parallel, we evaluated the performance of the iQ200 screen compared to that of Gram stain for sample quality. We screened 1,442 samples submitted for bacterial culture using the iQ200 analyzer; of these samples, 357 (24.8%) had a positive culture result. We identified the absence of microorganisms in the iQ200 screen as the strongest solitary predictor for a negative culture, with a sensitivity of 90.5% (323/357). The algorithm was further improved by performing logistic regression on leukocyte counts, which gave a cutoff of 65 leukocytes/µl to obtain the desired sensitivity of >95% (95.2%; 95% confidence interval [CI], 92.5 to 97.0), a negative predictive value of 97.3% (95% CI, 95.7 to 98.3), and an anticipated culture workload reduction of 44% (95% CI, 41 to 46). Concordance between sample quality based on Gram stain and iQ200 screening was only 72%, which was probably a result of interobserver effect in evaluation of the Gram stain. In conclusion, in our setting, screening by iQ200 proved to be a safe and cost-effective means to provide faster culture results, and it has the added benefit of a more objective evaluation of sample quality.

Capillary-electrophoresis mass spectrometry for the detection of carbapenemases in (multi-)drug-resistant Gram-negative bacteria.

In a time in which the spread of multidrug resistant microorganisms is ever increasing, there is a need for fast and unequivocal identification of suspect organisms to supplement existing techniques in the clinical laboratory, especially in single bacterial colonies. Mass-spectrometry coupled with efficient peptide separation techniques offer great potential for identification of resistant-related proteins in complex microbiological samples in an unbiased manner. Here, we developed a capillary electrophoresis-electrospray ionization-tandem mass spectrometry CE-ESI-MS/MS bottom-up proteomics workflow for sensitive and specific peptide analysis with the emphasis on the identification of ß-lactamases (carbapenemases OXA-48 and KPC in particular) in bacterial species. For this purpose, tryptic peptides from whole cell lysates were analyzed by sheathless CE-ESI-MS/MS and proteins were identified after searching of the spectral data against bacterial protein databases. The CE-ESI-MS/MS workflow was first evaluated using a recombinant TEM-1 ß-lactamase, resulting in 68% of the amino acid sequence being covered by 20 different unique peptides. Subsequently, a resistant and susceptible Escherichia coli lab strain were analyzed and based on the observed ß-lactamase peptides, the two strains could easily be discriminated. Finally, the method was tested in an unbiased setup using a collection of in-house characterized OXA-48 (n = 17) and KPC (n = 10) clinical isolates. The developed CE-ESI-MS/MS method was able to identify the presence of OXA-48 and KPC in all of the carbapenemase positive samples, independent of species and degree of susceptibility. Four negative controls were tested and classified as negative by this method. Furthermore, a number of extended-spectrum beta-lactamases (ESBL) were identified in the same analyses, confirming the multiresistant character in 19 out of 27 clinical isolates. Importantly, the method performed equally well on protein lysates from single colonies. As such, it demonstrates CE-ESI-MS/MS as a potential next generation mass spectrometry platform within the clinical microbiology laboratory.

Clinical impact of RT-PCR for pediatric acute respiratory infections: a controlled clinical trial.

OBJECTIVE: Real-time polymerase chain reaction (RT-PCR) testing is a quick sensitive method for detecting respiratory pathogens. We evaluated the diagnostic yield of RT-PCR assays and measured the effect of rapid reporting on patient care. METHODS: In a controlled clinical trial, nasal wash specimens were obtained from patients <12 years of age with suspected acute respiratory infections. In addition to the standard hospital protocol, RT-PCR assays for 17 pathogens were performed. The RT-PCR results were communicated to the clinicians within 12 to 36 hours in the intervention group and after 4 weeks in the control group. RESULTS: A total of 583 patients were included (mean age: 8.1 months [range: 0-107.5 months]): 298 in the intervention group and 285 in the control group. Eighty-two percent of nasal wash specimens tested positive for ≥1 pathogen. Respiratory syncytial virus was the most frequently encountered (55%) pathogen. There were no significant differences between the groups with respect to hospital admissions (intervention group: 223 admissions; control group: 211 admissions; P = .825), length of hospital stay (mean ± SD: 3.68 ± 2.68 days [intervention group] and 3.96 ± 2.67 days [control group]; P = .178), or duration of antibiotic use (mean ± SD: 6.52 ± 2.15 days [intervention group] and 6.97 ± 2.86 days [control group]; P = .490), when antibiotic treatment had been initiated. CONCLUSIONS: RT-PCR testing has a high yield of viral diagnoses, but rapid communication does not lead to decreases in hospital admissions, shorter hospital stays, or less antibiotic use for children with acute respiratory infections.