Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Respiratory Viruses in Wastewater Compared with Clinical Samples, Leuven, Belgium.

In a 2-year study in Leuven, Belgium, we investigated the use of wastewater sampling to assess community spread of respiratory viruses. Comparison with the number of positive clinical samples demonstrated that wastewater data reflected circulation levels of typical seasonal respiratory viruses, such as influenza, respiratory syncytial virus, and enterovirus D68.

The value of point-of-care tests for the detection of SARS-CoV-2 RNA or antigen in bronchoalveolar lavage fluid.

BACKGROUND: Transmission of SARS-CoV-2 from donor to recipient is a clinically relevant risk for developing severe COVID-19 after lung transplantation (LTx). This risk of iatrogenic transmission can be reduced by timely detection of viral RNA or antigen in samples of bronchoalveolar lavage (BAL) fluid obtained at the time of lung procurement. We aimed to retrospectively evaluate the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples using three point-of-care tests (POCTs). METHODS: BAL fluid samples came from patients hospitalized in an intensive care unit during the COVID-19 pandemic. These pandemic samples were scored as positive or negative for SARS-CoV-2 by a RT-qPCR comparator assay for orf1ab. Three commercially available POCTs were then evaluated: cobas SARS-CoV-2 & Influenza A/B assay with the cobas Liat RT-qPCR system (Roche Diagnostics), ID NOW COVID-19 and COVID-19 2.0 (Abbott), and SARS-CoV-2 Rapid Antigen Test (RAT) (Roche Diagnostics). Samples from the pre-pandemic era served as negative controls. RESULTS: We analyzed a total of 98 BAL fluid samples, each from a different patient: 58 positive pandemic samples (orf1ab Ct<38), 20 putatively negative pandemic samples (orf1ab Ct≥38), and 20 pre-pandemic samples. Univariate logistic regression shows that the probability of detection was highest for cobas Liat, followed by ID NOW, and then RAT. Of clinical relevance, cobas Liat detected SARS-CoV-2 RNA in 30 of the 31 positive pandemic samples that were collected within 10 days after RT-qPCR diagnosis of SARS-CoV-2 infection. None of the 20 pre-pandemic samples had a false-positive result for any POCT. CONCLUSIONS: POCTs enable the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples and may provide additional information to decide if donor lungs are suitable for transplantation. Detection of respiratory pathogens with POCTs at the time of donor lung procurement is a potential strategy to increase safety in LTx by preventing iatrogenic transmission and severe postoperative infections.

An observational cohort study of histological screening for BK polyomavirus nephropathy following viral replication in plasma.

Systematic screening for BKPyV-DNAemia has been advocated to aid prevention and treatment of polyomavirus associated nephropathy (PyVAN), an important cause of kidney graft failure. The added value of performing a biopsy at time of BKPyV-DNAemia, to distinguish presumptive PyVAN (negative SV40 immunohistochemistry) and proven PyVAN (positive SV40) has not been established. Therefore, we studied an unselected cohort of 950 transplantations, performed between 2008-2017. BKPyV-DNAemia was detected in 250 (26.3%) transplant recipients, and positive SV40 in 91 cases (9.6%). Among 209 patients with a concurrent biopsy at time of first BKPyV-DNAemia, 60 (28.7%) biopsies were SV40 positive. Plasma viral load showed high diagnostic value for concurrent SV40 positivity (ROC-AUC 0.950, 95% confidence interval 0.916-0.978) and the semiquantitatively scored percentage of tubules with evidence of polyomavirus replication (pvl score) (0.979, 0.968-0.988). SV40 positivity was highly unlikely when plasma viral load is below 4 log10 copies/ml (negative predictive value 0.989, 0.979-0.994). In SV40 positive patients, higher plasma BKPyV-DNA load and higher pvl scores were associated with slower viral clearance from the blood (hazard ratio 0.712, 95% confidence interval 0.604-0.839, and 0.327, 0.161-0.668, respectively), whereas the dichotomy positivity/negativity of SV40 immunohistochemistry did not predict viral clearance. Although the pvl score offers some prognostic value for viral clearance on top of plasma viral load, the latter provided good guidance for when a biopsy was unnecessary to exclude PyVAN. Thus, the distinction between presumptive and proven PyVAN, based on SV40 immunohistochemistry, has limited clinical value. Hence, management of BKPyV-DNAemia and immunosuppression reduction should be weighed against the risk of occurrence of rejection, or exacerbation of rejection observed concomitantly.

Indoor air surveillance and factors associated with respiratory pathogen detection in community settings in Belgium.

Currently, the real-life impact of indoor climate, human behaviour, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings in Belgium for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. Pathogen detection and concentration varied significantly by pathogen, month, and age group in generalised linear (mixed) models and generalised estimating equations. High CO2 and low natural ventilation were independent risk factors for detection. The odds ratio for detection was 1.09 (95% CI 1.03-1.15) per 100 parts per million (ppm) increase in CO2, and 0.88 (95% CI 0.80-0.97) per stepwise increase in natural ventilation (on a Likert scale). CO2 concentration and portable air filtration were independently associated with pathogen concentration. Each 100ppm increase in CO2 was associated with a qPCR Ct value decrease of 0.08 (95% CI -0.12 to -0.04), and portable air filtration with a 0.58 (95% CI 0.25-0.91) increase. The effects of occupancy, sampling duration, mask wearing, vocalisation, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.

Blood Mucorales PCR to track down Aspergillus and Mucorales co-infections in at-risk hematology patients: A case-control study.

Introduction: Serum Mucorales PCR can precede the final diagnosis of invasive mucormycosis by several days or weeks and could therefore be useful as a non-invasive screening tool. Methods: We assessed the performance of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics, Maastricht, The Netherlands) on prospectively collected banked sera from hematology patients at risk for invasive mould infections. We evaluated if there is an underestimated incidence of missed Mucorales co-infections in patients with invasive aspergillosis (IA). We tested Mucorales PCR on the sera of all patients with a diagnosis of at least possible IA (EORTC-MSGERC consensus criteria) before the start of any antifungal therapy, and in a control group of similar high-risk hematology patients without IA (in a 1:4 ratio). When a positive Mucorales PCR was observed, at least 5 serum samples taken before and after the positive one were selected. Results: Mucorales PCR was performed in 46 diagnostic serum samples of cases and in 184 controls. Serum Mucorales PCR was positive in 4 cases of IA (8.7%; 12.9% of probable cases) and in 1 control case (0.5%) (p=0.0061, OR=17.43 (1.90-159.96). Post-mortem cultures of the positive control became positive for Rhizopus arrhizus. Mortality of IA cases with and without a positive Mucorales PCR was not significantly different. Only in the PCR positive control case, serial serum samples before and after the diagnostic sample were also positive. Discussion: It is not entirely clear what a positive Mucorales PCR in these cases implies since the 4 Mucorales PCR positive cases were treated with antifungals with activity against Mucorales. In addition, PCR was positive only once. This study does not provide enough evidence to implement Mucorales PCR screening. However, our findings emphasize once more the importance of considering the possibility of dual mould infections, even in patients with a positive galactomannan detection.

Full-genome next-generation sequencing of hepatitis C virus to assess the accuracy of genotyping by the commercial assay LiPA and the prevalence of resistance-associated substitutions in a Belgian cohort.

BACKGROUND: Although most currently used regimens for Hepatitis C virus (HCV) infections can be initiated without prior knowledge of genotype and subtype, genotyping is still useful to identify patients who might benefit from a personalized treatment due to resistance to direct-acting antivirals (DAA). OBJECTIVES: To assess the utility of full-genome next-generation sequencing (FG-NGS) for HCV genotyping. STUDY DESIGN: 138 HCV plasma samples previously genotyped by VERSANT HCV Genotype Assay (LiPA) were subjected to FG-NGS and phylogenetically genotyped Genome Detective. Consensuses were analysed by HCV-GLUE for resistance-associated substitutions (RASs) and their impact on treatment response was investigated. RESULTS: 102/138 (73.9%) samples were sequenced to a genome coverage and depth of >90% of the HCV open reading frame covered by >100 reads/site. Concordant genotype and subtype results were assigned in 97.1% and 79.4% of samples, respectively. FG-NGS resolved the subtype of 13.7% samples that had ambiguous calls by LiPA and identified one dual infection and one recombinant strain. At least one RAS was found for the HCV genes NS3, NS5A, and NS5B in 2.91%, 36.98% and 27.3% samples, respectively. Irrespective of the observed RAS, all patients responded well to DAA treatment, except for HCV1b-infected patients treated with Zepatier (33.3% failure rate (5/15)). CONCLUSION: While LiPA and FG-NGS showed overall good concordance, FG-NGS improved specificity for subtypes, recombinant and mixed infections. FG-NGS enabled the detection of RAS, but its predictive value for treatment outcome in DAA-naïve patients remains uncertain. With additional refinements, FG-NGS may be the way forward for HCV genotyping.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

No SARS-CoV-2 carriage observed in children attending daycare centers during the intial weeks of the epidemic in Belgium.

To gain knowledge about the role of young children attending daycare in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic, a random sample of children (n = 84) aged between 6 and 30 months attending daycare in Belgium was studied shortly after the start of the epidemic (February 29th) and before the lockdown (March 18th) by performing in-house SARS-CoV-2 real-time polymerase chain reaction. No asymptomatic carriage of SARS-CoV-2 was detected, whereas common cold symptoms were common (51.2%). Our study shows that in Belgium, there was no sign of early introduction into daycare centers at the moment children being not yet isolated at home, although the virus was clearly circulating. It is clear that more evidence is needed to understand the actual role of young children in the transmission of SARS-CoV-2 and their infection risk when attending daycare.

Laboratory information system requirements to manage the COVID-19 pandemic: A report from the Belgian national reference testing center.

OBJECTIVE: The study sought to describe the development, implementation, and requirements of laboratory information system (LIS) functionality to manage test ordering, registration, sample flow, and result reporting during the coronavirus disease 2019 (COVID-19) pandemic. MATERIALS AND METHODS: Our large (>12 000 000 tests/y) academic hospital laboratory is the Belgian National Reference Center for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We have performed a moving total of >25 000 SARS-CoV-2 polymerase chain reaction tests in parallel to standard routine testing since the start of the outbreak. A LIS implementation team dedicated to develop tools to remove the bottlenecks, primarily situated in the pre- and postanalytical phases, was established early in the crisis. RESULTS: We outline the design, implementation, and requirements of LIS functionality related to managing increased test demand during the COVID-19 crisis, including tools for test ordering, standardized order sets integrated into a computerized provider order entry module, notifications on shipping requirements, automated triaging based on digital metadata forms, and the establishment of databases with contact details of other laboratories and primary care physicians to enable automated reporting. We also describe our approach to data mining and reporting of actionable daily summary statistics to governing bodies and other policymakers. CONCLUSIONS: Rapidly developed, agile extendable LIS functionality and its meaningful use alleviates the administrative burden on laboratory personnel and improves turnaround time of SARS-CoV-2 testing. It will be important to maintain an environment that is conducive for the rapid adoption of meaningful LIS tools after the COVID-19 crisis.

Serial Detection of Circulating Mucorales DNA in Invasive Mucormycosis: A Retrospective Multicenter Evaluation.

Invasive mucormycosis is a fungal infection with high mortality. Early diagnosis and initiation of appropriate treatment is essential to improve survival. However, current diagnostic tools suffer from low sensitivity, leading to delayed or missed diagnoses. Recently, several PCR assays for the detection of Mucorales DNA have been developed. We retrospectively assessed the diagnostic and kinetic properties of a commercial Mucorales PCR assay (MucorGenius, PathoNostics) on serial blood samples from patients with culture-positive invasive mucormycosis and found an overall sensitivity of 75%. Importantly, a positive test preceded a positive culture result by up to 81 days (median eight days, inter-quartile range 1.75-16.25). After initiation of appropriate therapy, the average levels of circulating DNA decreased after one week and stabilized after two weeks. In conclusion, detection of circulating Mucorales DNA appears to be a good, fast diagnostic test that often precedes the final diagnosis by several days to weeks. This test could be especially useful in cases in which sampling for culture or histopathology is not feasible.

A decade of enterovirus genetic diversity in Belgium.

BACKGROUND: Enteroviruses are responsible for a wide range of clinical symptoms.Enterovirus D68 was already known to cause mild to severe respiratory infections, but in the last few years, it has also been associated with neurological symptoms and acute flaccid paralysis. OBJECTIVES: In this epidemiological surveillance in Belgium, 1521 enteroviruspositive samples were genotyped. STUDY DESIGN: Enterovirus-positive patient samples were collected from the University Hospitals Leuven and other hospitals and medical practices in Belgium from 2007 to 2018. Molecular typing was done by RT-PCR using different primers sets. EV-A and EV-B were typed by sequencing part of VP1. For EVC and EV-D, the VP4/VP2 region was used together with the non-coding region. RESULTS: In this epidemiological survey with samples collected over 12 years, 35 different EV types were detected in 1521 patient samples. Enterovirus species B was by far the most dominant species in our samples (93%). Echovirus 30 was most frequently found (24%), followed by echovirus 6 (8%) and echovirus 9 (7%). In 2018, there was an outbreak for the first time of enterovirus D68 with severe respiratory infections but no acute flaccid paralysis. Phylogenetic analyses showed that the collected outbreak strains coexist in different clades. CONCLUSIONS: For more than a decade, the circulating enterovirus strains were investigated in Belgium. During this time span, echovirus 30 was the most frequently detected and peaked every 3 years. Enterovirus D68 began an upsurge in 2018, but thus far without being clinically associated with acute flaccid paralysis.

Predictive value of JC virus PCR in cerebrospinal fluid in the diagnosis of PML.

OBJECTIVE: To assess the predictive value of JC virus (JCV) PCR in cerebrospinal fluid (CSF) in the diagnosis of progressive multifocal leukoencephalopathy (PML). METHODS: We conducted a retrospective database query to identify patients with positive CSF JCV PCR. Clinical features, final diagnosis and quantitative PCR results were obtained. RESULTS: A positive CSF JCV PCR had a PPV of 10.4% for the diagnosis of PML. A weakly positive PCR had a PPV of 1.6%, whereas a moderately to highly positive PCR had a PPV of 92.3%. A PPV of 0.0% was observed in immunocompetent patients and in patients without compatible clinical or radiological features. CONCLUSIONS: A false-positive CSF JCV PCR is highly prevalent in our clinical practice. This test should be reserved for patients with a clinical suspicion of PML and the quantitative result of the PCR should be taken into account when making the diagnosis of PML.

Migrating a lab-developed MERS-CoV real-time PCR to 3 "Sample to Result" systems: experiences on optimization and validation.

The goal of the study was to adapt our Middle East respiratory syndrome coronavirus (MERS-CoV) lab-developed test (LDT) to 3 "Sample to Result" (S2R) systems: BD MAX (BD), ELITe InGenius (ELITechGroup), and ARIES (Luminex). The BD MAX and InGenius system allowed use of lab-developed primers and TaqMan probes, while ARIES required conversion to MultiCode primers for melting curve analysis. Each device required ≤1 day of training and assay optimization. No discordant results were noted after analysis of 32 External Quality Control (EQC) samples. On a 10-fold dilution series of a MERS-CoV-positive EQC sample, InGenius obtained the highest detection rate. Laboratory technicians rated the ARIES as the user-friendliest. It also required the least hands-on time. BD MAX had the lowest turnaround time and highest throughput. While each device had distinguishing system properties with associated (dis)advantages, the 3 S2R systems were comparable in terms of assay development and validation.

Beta-d-Glucan for Diagnosing Pneumocystis Pneumonia: a Direct Comparison between the Wako ß-Glucan Assay and the Fungitell Assay.

Measuring serum beta-d-glucan (BDG) is a useful tool for supporting a quantitative PCR (qPCR)-based diagnosis of suspected Pneumocystis pneumonia (PCP) with bronchoalveolar lavage (BAL) fluid. Since the 2000s, the Fungitell assay was the only BDG assay which was FDA cleared and Conformité Européenne (CE) marked. However, the Wako ß-glucan test was also recently CE marked and commercialized. We analyzed archived sera from 116 PCP cases (who were considered to have PCP based on compatible clinical and radiological findings plus a BAL fluid qPCR threshold cycle value of ≤28) and 114 controls (those with a BAL fluid qPCR threshold cycle value of >45 and no invasive fungal infection) using the Fungitell and Wako assays in parallel and assessed their diagnostic performance using the manufacturer's proposed cutoffs of 80 pg/ml and 11 pg/ml, respectively. We found the Wako assay to be more specific (0.98 versus 0.87, P < 0.001) and the Fungitell assay to be more sensitive (0.78 versus 0.85, P = 0.039) at the proposed cutoffs. Overall performance, as determined by the area under the receiver operating characteristic curve, was similar for both assays. We determined a new Wako assay cutoff (3.616 pg/ml) to match the sensitivity of the Fungitell assay (0.88 at a cutoff of ≥60 pg/ml). Using this newly proposed cutoff, the specificity of the Wako assay was significantly better than that of the Fungitell assay (0.89 versus 0.82, P = 0.011). In conclusion, the Wako assay performed excellently compared to the Fungitell assay for the diagnosis of presumed PCP based on qPCR. In addition, contrary to the Fungitell assay, the Wako assay allows for single-sample testing with lower inter- and intrarun variability. Finally, we propose an optimized cutoff for the Wako assay to reliably exclude PCP.

Diagnostic accuracy of digital RNA quantification versus real-time PCR for the detection of respiratory syncytial virus in nasopharyngeal aspirates from children with acute respiratory infection.

BACKGROUND: Virus-specific molecular assays such as real-time polymerase chain reaction (RT-PCR) are regularly used as the gold standard to diagnose viral respiratory tract infections, but simultaneous detection of multiple different pathogens is often challenging. A multiplex digital method of RNA quantification, nCounter (NanoString Technologies), can overcome this disadvantage and identify, in a single reaction, the presence of different respiratory viruses. OBJECTIVES: To evaluate the accuracy of nCounter to identify and quantify RSV-A and RSV-B in nasopharyngeal aspirates (NPA) of children (6-23-months-old) with acute respiratory infection. STUDY DESIGN: NPA was collected at enrolment in a prospective cross-sectional study conducted in Salvador, Brazil. A quantitative RT-PCR with a subgroup-specific primer and probeset for RSV-A and RSV-B was performed in parallel with a customized nCounter probeset containing viral targets in NPA. RESULTS: Of 559 NPA tested, RSV was detected by RT-PCR in 139 (24.9%), by nCounter in 122 (21.8%) and by any method in 158 (28.3%) cases. Compared to the gold standard of qRT-PCR, sensitivity of nCounter was 74.3% (95%CI:63.3%-82.9% RSV-A) and 77.6% (95%CI:66.3%-85.9% RSV-B); specificity was 98.4% (95%CI:96.8%-99.2% RSV-A) and 97.8% (95%CI:96.0%-98.8% RSV-B); positive predictive value was 87.3% (95%CI:76.9%-93.4% RSV-A) and 82.5% (95%CI:71.4%-90.0% RSV-B) and negative predictive value was 96.1% (95%CI:94.1%-97.5% RSV-A), and 96.9% (95%CI:95.1%-98.2% RSV-B). Accuracy was 95.2% (95%CI:93.1%-96.7%) for RSV-A and 95.3% (95%CI:93.3%-96.9%) for RSV-B, while both methods significantly correlated for RSV-A (r = 0.44, p = 8 × 10-5) and RSV-B (r = 0.73, p = 3 × 10-12) quantification. CONCLUSIONS: nCounter is highly accurate in detecting RSV-A/B in NPA. Robustness and high-throughput multiplexing indicate its use in large-scale epidemiological studies.

Prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in Belgium.

BACKGROUND: Acute Respiratory Infections (ARIs) are a major health problem, especially in young children and the elderly. OBJECTIVES: Insights into the seasonality of respiratory viruses can help us understand when the burden on society is highest and which age groups are most vulnerable. STUDY DESIGN: We monitored six respiratory viruses during five consecutive seasons (2011-2016) in Belgium. Patient specimens (n=22876), tested for one or more of the following respiratory viruses, were included in this analysis: Influenza viruses (IAV & IBV), Human respiratory syncytial virus (hRSV), Human metapneumovirus (hMPV), Adenovirus (ADV) and Human parainfluenza virus (hPIV). Data were analysed for four age categories: <6y, 6-17y, 18-64y and ≥65y. RESULTS: Children <6y had the highest infection rates (39% positive vs. 20% positive adults) and the highest frequency of co-infections. hRSV (28%) and IAV (32%) caused the most common respiratory viral infections and followed, like hMPV, a seasonal pattern with winter peaks. hRSV followed an annual pattern with two peaks: first in young children and ±7 weeks later in elderly. This phenomenon has not been described in literature so far. hPIV and ADV occurred throughout the year with higher rates in winter. CONCLUSIONS: Children <6y are most vulnerable for respiratory viral infections and have a higher risk for co-infections. hRSV and IAV are the most common respiratory infections with peaks during the winter season in Belgium.

Comparison of PCR-Electrospray Ionization Mass Spectrometry with 16S rRNA PCR and Amplicon Sequencing for Detection of Bacteria in Excised Heart Valves.

Identification of the causative pathogen of infective endocarditis (IE) is crucial for adequate management and therapy. A broad-range PCR-electrospray ionization mass spectrometry (PCR-ESI-MS) technique was compared with broad-spectrum 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) for the detection of bacterial pathogens in 40 heart valves obtained from 34 definite infective endocarditis patients according to the modified Duke criteria and six nonendocarditis patients. Concordance between the two molecular techniques was 98% for being positive or negative, 97% for concordant identification up to the genus level, and 77% for concordant identification up to the species level. Sensitivity for detecting the causative pathogen (up to the genus level) in excised heart valves was 88% for 16S rRNA PCR and 85% for PCR-ESI-MS; the specificity was 83% for both methods. The two molecular techniques were significantly more sensitive than valve culture (18%) and accurately identified bacteria in excised heart valves. In eight patients with culture-negative IE, the following results were obtained: concordant detection of Coxiella burnetii (n = 2), Streptococcus gallolyticus (n = 1), Propionibacterium acnes (n = 1), and viridans group streptococci (n = 1) by both molecular tests, detection of P. acnes by PCR-ESI-MS whereas the 16S rRNA PCR was negative (n = 1), and a false-negative result by both molecular techniques (n = 2). In one case of IE caused by viridans streptococci, PCR-ESI-MS was positive for Enterococcus spp. The advantages of PCR-ESI-MS compared to 16S rRNA PCR are its automated workflow and shorter turnaround times.

Epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital.

BACKGROUND: The prevalence of respiratory viruses in adults is largely underexplored, as most studies focus on children. Additionally, in severely ill or immunocompromised adults, where respiratory infections are mostly attributed to bacteria and fungi; respiratory viruses can lead to severe complications. OBJECTIVES: To evaluate the epidemiology of respiratory viruses in bronchoalveolar lavage fluid (BAL) specimens from patients with lower respiratory tract disease. The study population consisted of different groups including immunocompetent patients (control patients), solid organ transplant recipients, patients with haematological malignancies and other immunocompromised adults. STUDY DESIGN: A total of 134 BAL fluid specimens collected during 2009-2011 were retrospectively assessed with the new commercial multiplex real-time PCR FTD Respiratory 21 Plus(®), targeting 18 different viruses and 2 atypical bacterial pathogens. RESULTS: Viral or atypical bacterial pathogens were detected in 29.1% of BAL fluid specimens. Coronaviruses were most prevalent (13.4%), followed by rhinoviruses (5.2%), RSV (4.5%) and bocaviruses (3.7%). Comparing the total number of viruses detected, a statistically significant difference was observed between the control group and patients with haematological malignancies (27.5% vs. 57.1%, p<0.05). CONCLUSION: In conclusion, our study highlights the high prevalence of respiratory viruses in BAL fluid specimens from adult patients with lower respiratory tract disease. The methods to be used should be sensitive and cover a wide range of potential pathogens. The specific patient population can also influence the detection rates of respiratory viruses.