Implementation of full-length 16S nanopore sequencing for bacterial identification in a clinical diagnostic setting.

This study describes the implementation of 16S nanopore sequencing in a diagnostic lab for pathogen identification without prior enrichment. First, the universality of the test and taxonomic resolution was evaluated for 78 clinically relevant bacteria (69 known and 9 unknown bacterial cultures). Next, the diagnostic value of the test was evaluated based on clinical samples. It was shown that 16S sequencing can be used both for identification of unknown cultures and to find bacteria directly in the clinical sample without cultivation. All culture-positive samples (n=11) tested positive with 16S sequencing directly performed on the sample, but bacteria were found as well in 15/30 culture-negative samples. Pathogenic bacteria were found in a background of commensal flora, and even complex polymicrobial infections could be unraveled. This study demonstrates the feasibility of implementing 16S nanopore sequencing in a clinical diagnostic setting and demonstrates its value for the diagnosis of culture-negative and polymicrobial infections.

Multicenter evaluation of the FilmArray Meningitis/Encephalitis assay in a routine setting.

Introduction. The FilmArray Meningitis/Encephalitis (FA-ME) Panel (Biofire, Salt Lake City, Utah, US) enables fast and automated detection of 14 pathogens in cerebrospinal fluid (CSF).Gap statement. The performance of the FA-ME panel in a real routine setting has not yet been described and could lead to better patient management in cases of good performance.Aim. This multicenter study verified the FA-ME panel analytical performance in a routine hospital setting.Methodology. Between April 2016 and April 2018, 454 CSF samples were analysed with the FA-ME panel and compared with routine diagnostics. In cases of discrepancy or lack of a comparator result, a profound analysis based on patient records and other laboratory results was performed.Results. A first analysis of 65 frozen samples, suspicious for meningitis had a 89 % concordance with routine diagnostics. The limit of detection (LOD) was confirmed for all pathogens except for Streptococcus agalactiae and a strain of Haemophilus influenzae (Escherichia coli K1 and Cryptococcus gattii LOD experiments were not performed). The routine evaluation showed a positive result in 114 (25 %) clinical samples for at least one target. In three samples co-infections were found. After discrepancy analysis, overall sensitivity was 98 % (false negative FA-ME results for one HSV2, two HSV1 and two parechovirus). Four FA-ME results were considered false positive (two HHV6, one VZV and one E. coli K1), resulting in an overall specificity of >99 %. A clinical added value of the assay was seen in the diagnosis of eight cases of bacterial meningitis.Conclusion. Because of its rapidity and ease of use, the FA-ME panel has great potential in the diagnosis of central nervous infections. Implementation can improve clinical management, but costs and analytical limitations need to be addressed to convince clinicians and laboratories of its value.

Multicenter study of automated systems for colistin susceptibility testing.

PURPOSE: Broth microdilution (BMD) stays as the reference testing method for determination of antimicrobial susceptibility testing (AST) to colistin and is considered essential for patient management and for monitoring of colistin resistance. This multicenter study aimed to evaluate the performance of automated systems for colistin AST among Enterobacterales as an alternative for BMD since the majority of laboratories use automated systems as first-line method. METHODS: Twenty colistin resistant (COL-R) including 10 MCR producers and 10 colistin-susceptible (COL-S) Enterobacterales isolates were blindly tested for colistin susceptibility with the routine automated AST systems used by 8 laboratories (3 with BD Phoenix, 3 with Vitek2 and 2 with MicroScan). Additionally, 3 reference strains (E. coli ATCC 25922, E. coli NCTC 13846, and one COL-R mcr-negative K. pneumoniae M/14750) were tested in triplicate by each laboratory. RESULTS AND CONCLUSION: Results were compared with BMD performed at the reference laboratory. BD Phoenix and MicroScan automated AST systems provide accurate and reproducible categorical results for the testing of colistin in Enterobacterales. However, Vitek2 system showed poor performance for the detection of COL-R isolates especially those with MICs close to the susceptibility breakpoint (categorical agreement of 88% and precision categorical agreement of 81%).

Infection prevention and control challenges in Flemish homecare nursing: a pilot study.

Home nursing is evolving towards more invasive care. Nevertheless, no national data are available on the prevalence of HAI in this setting. The aim of this pilot study is to explore the Flemish home care setting as a first step toward a national surveillance program. A survey, focused on patient characteristics and HAI, was conducted between 7 May and 20 July 2018 on 711 Flemish patients. Most of the patients (74%) are 65 years or older and half of them had a form of comorbidity. Assisting with personal hygiene and wound care were the most frequent services delivered by home care nurses. A comparison of the prevalence of infections diagnosed by a physician or applying uniform criteria (ECDC), revealed a similar prevalence of skin and soft tissue infections (9% vs. 8.5%) and urinary tract infections (4% vs. 4.5%). A positive MDRO-screening was found in 6% of the patients. This pilot study is a first step towards a standardized national surveillance in home care to collect information on the prevalence of HAI and it reveals several interesting facts and study pitfalls for this setting.

Human bocavirus infection in Belgian children with respiratory tract disease.

Human bocavirus (HBoV) has been detected primarily in children with acute lower respiratory tract disease (LRTD), but its occurrence, clinical profile, and role as a causative agent of RTD are not clear. The aim of this study was to investigate the prevalence and the potential clinical relevance of HBoV. Using molecular tests, we tested 1352 nasopharyngeal samples obtained between October 1, 2017 and April 30, 2018 from children up to the age of 16 with RTD for the presence of HBoV DNA and 20 other respiratory pathogens at three different hospitals in Belgium. HBoV was detected in 77 children with a median age of 10.6 months. Consecutive samples were available for 15 HBoV-positive children and showed persistent HBoV positivity in four of them. Monoinfection was observed in six infants. Four of them were born prematurely and were infected during hospitalization at the neonatal intensive care unit (NICU). Only one of these six monoinfected children was diagnosed with recurrent wheezing due to HBoV. This child was carried to term and had a high viral load. Coinfections, most frequently with rhinovirus (52.1%) and adenovirus (49.3%), were observed in 72 patients. In seventeen of them in which HBoV was present at high viral load or higher viral load than its copathogens, bronchi(oli)tis (n = 8), recurrent wheezing (n = 8) or episodic wheezing (n = 1) were diagnosed. Our results suggest that HBoV infection at high viral load in infants is associated with wheezing (P = 0.013, Cramer's V = 0.613).

A case of nosocomial Legionnaires' disease: The importance of early diagnosis, adequate prevention and eradication measures.

This case-report describes a patient with confirmed nosocomial Legionnaires' disease, a diagnosis which should be suspected when pneumonia does not respond to empiric therapy with beta-lactam antibiotics, or develops in the presence of certain risk factors. Culture is currently the golden standard for diagnosis, although the use of more modern techniques, such as a combination of urinary antigen testing and polymerase chain reaction, are on the rise. Specific detection and eradication methods are discussed.

Implementation of real-time RT-PCR for detection of human metapneumovirus and its comparison with enzyme immunoassay.

Human metapneumovirus (hMPV) is responsible for outbreaks of bronchiolitis in winter and early spring in young children. Due to the relatively recent discovery of hMPV, the diagnostic opportunities are limited, while differential diagnosis with respiratory syncytial virus (RSV) remains important. We validated the RT-PCR by comparing various methods of RNA extraction, one-step RT-PCR kits and primer-probe combinations. The optimized RT-PCR was evaluated using 47 nasopharyngeal aspirates (NPAs) collected from children younger than 5 years, with clinically suspected RSV infection. The evaluated RT-PCRs were also compared to a commercially available hMPV enzyme immunoassay (EIA). We found 8.5% hMPV positivity with both RT-PCRs, in agreement with published literature. hMPV EIA showed positive and indeterminate results in 17% and 8.5%, respectively, of the tested NPAs. Positive RT-PCR samples were positive or indeterminate by hMPV EIA. Samples that were positive for RSV and influenza A virus interfered with the hMPV EIA. In conclusion, although RT-PCR is already a valuable tool for diagnosing hMPV infections, further optimization of the RT-PCR method is recommended. The hMPV EIA kit shows poor specificity and therefore needs further improvement.

Analytical interferences in point-of-care testing glucometers by icodextrin and its metabolites: an overview.

Current point-of-care testing (POCT) glucometers are based on various test principles. Two major method groups dominate the market: glucose oxidase-based systems and glucose dehydrogenase-based systems using pyrroloquinoline quinone (GDH-PQQ) as a cofactor. The GDH-PQQ-based glucometers are replacing the older glucose oxidase-based systems because of their lower sensitivity for oxygen. On the other hand, the GDH-PQQ test method results in falsely elevated blood glucose levels in peritoneal dialysis patients receiving solutions containing icodextrin (e.g., Extraneal; Baxter, Brussels, Belgium). Icodextrin is metabolized in the systemic circulation into different glucose polymers, but mainly maltose, which interferes with the GDH-PQQ-based method. Clinicians should be aware of this analytical interference. The POCT glucometers based on the GDH-PQQ method should preferably not be used in this high-risk population and POCT glucose results inconsistent with clinical suspicion of hypoglycemic coma should be retested with another testing system.

Critical evaluation of connectivity-based point of care testing systems of glucose in a hospital environment.

BACKGROUND: In recent years a number of point of care testing (POCT) glucometers were introduced on the market. We investigated the analytical variability (lot-to-lot variation, calibration error, inter-instrument and inter-operator variability) of glucose POCT systems in a university hospital environment and compared these results with the analytical needs required for tight glucose monitoring. METHODS: The reference hexokinase method was compared to different POCT systems based on glucose oxidase (blood gas instruments) or glucose dehydrogenase (handheld glucometers). Based upon daily internal quality control data, total errors were calculated for the various glucose methods and the analytical variability of the glucometers was estimated. RESULTS: The total error of the glucometers exceeded by far the desirable analytical specifications (based on a biological variability model). Lot-to-lot variation, inter-instrument variation and inter-operator variability contributed approximately equally to total variance. As in a hospital environment, distribution of hematocrit values is broad, converting blood glucose into plasma values using a fixed factor further increases variance. The percentage of outliers exceeded the ISO 15197 criteria in a broad glucose concentration range. CONCLUSIONS: Total analytical variation of handheld glucometers is larger than expected. Clinicians should be aware that the variability of glucose measurements obtained by blood gas instruments is lower than results obtained with handheld glucometers on capillary blood.