Kurt Beuselinck

Polyomavirus infection in pediatric renal transplant recipients: Evaluation using a quantitative real-time PCR technique

Abstract:  Polyomavirus infection and related nephropathy is being increasingly recognized as an important cause of allograft dysfunction in adult renal transplant recipients. We prospectively monitored pediatric renal transplant recipients for the presence of BK and JC polyomavirus in urine and blood using a quantitative PCR assay to evaluate the prevalence and clinical relevance of polyomavirus infection in the pediatric renal transplant population. Of 46 pediatric renal recipients who were evaluated, nine (20%) demonstrated isolated BKV viruria, while five (11%) had concomitant BKV viremia and viruria. JCV viruria was found in eight (17%) patients. BKV viremia was associated with a significantly higher urinary BKV viral load: median urinary viral load 1.9 × 109 copies/mL (range 6.7 × 102–1.8 × 1011) for the group with concomitant viremia and viruria vs. 1.8 × 103 copies/mL (range 2.5 × 102–4.5 × 106) for the group with isolated viruria (p < 0.0001). In children that were followed prospectively since their transplantation, the BKV urinary viral load increased markedly before viremia became detectable a few weeks later. None of the patients with JCV viruria or isolated BKV viruria had renal dysfunction. Among the five patients with BKV in both urine and blood, two developed biopsy‐proven BKV nephropathy associated with deterioration of the renal function. Management of the BKV nephropathy consisted of reduction of immunosuppression alone or in combination with antiviral treatment with cidofovir. This study shows that polyomavirus infection and related interstitial nephritis is a relevant clinical issue in the pediatric renal transplant population. Monitoring the polyomaviral load in the urine and the blood of the patients using a quantitative PCR technique is a useful tool in the diagnosis and subsequent management of this infection. Even before viremia is present, an important rise in the urinary viral load should draw the attention of the transplant clinician and raise the issue of adapting the immunosuppression.

Clinical Predictive Value of Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples

The aim of this study was to evaluate the diagnostic reliability and prognostic significance of the quantification of cytomegalovirus (CMV) DNA in amniotic fluid (AF). We retrospectively reviewed the results for 282 amniotic fluid samples that had been tested for CMV by a quantitative real-time PCR. We observed three cases in which no CMV genomes were detected in the AF but in which the children were nevertheless congenitally infected. Hence, we conclude that a negative result by PCR for CMV in AF cannot rule out the possibility of congenital infection. No false-positive PCR results were observed. A correlation between the CMV viral load in AF and the fetal and neonatal outcomes could not be demonstrated in our study. Instead, a correlation was found between the CMV viral load and the gestational age at the time of amniocentesis.

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

Abstract 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.

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

ABSTRACT 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.

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.

Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study

OBJECTIVES For adequate management and therapy of infective endocarditis (IE), identification of the causative pathogen is crucial but molecular testing results are not currently included in diagnostic criteria. The added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing (16S rRNA PCR) performed on excised heart valves from patients with IE was evaluated alongside the effect of pre-operative antibiotics on the performance of blood culture (BC), valve culture (VC) and 16S rRNA PCR. METHODS All patients undergoing valve surgery for definite or possible IE, according to modified Duke Criteria, were prospectively included from July 2013 up to and including June 2016. RESULTS In all, 127 patients were included. Sensitivity for detecting the causative micro-organism in 120 post-operative definite IE patients was 26% for VC and 87% for BC and 16S rRNA PCR. 16S rRNA PCR, VC and BC were equally sensitive for different valve types and causative pathogens. In 27 (21%) definite IE patients, 16S rRNA PCR clarified discrepant culture results or was the only method identifying the causative pathogen. In 12 (10%) post-operative definite IE cases, molecular testing results influenced antimicrobial therapy. CONCLUSIONS The very good performance characteristics, added diagnostic value and impact on antimicrobial therapy of molecular testing of heart valves should support the incorporation of molecular testing in diagnostic criteria and guidelines for IE.

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.