An original multiplex method to assess five different SARS-CoV-2 antibodies.

OBJECTIVES: Accurate SARS-CoV-2 serological assays are urgently needed to help diagnose infection, determine past exposure of populations and assess the response to future vaccines. The study aims at assessing the performance of the multiplex D-tek COVIDOT 5 IgG assay for the detection of SARS-CoV-2 IgG antibodies (N, S1+S2, S1, S2 and RBD). METHODS: Sensitivity and dynamic trend to seropositivity were evaluated in 218 samples obtained from 46 rRT-PCR confirmed COVID-19 patients. Non-SARS-CoV-2 sera (n=118) collected before the COVID-19 pandemic with a potential cross-reaction to the SARS-CoV-2 immunoassay were included in the specificity analysis. RESULTS: A gradual dynamic trend since symptom onset was observed for all IgG antibodies. Sensitivities before day 14 were suboptimal. At ≥21 days, sensitivities reached 100% (93.4-100%) for N, S1+S2, S2 and RBD-directed IgG and 96.3% (87.3-99.6%) for S1-directed IgG. In 42 out of 46 patients (91.3%), all five antibodies were detected at ≥14 days. The four remaining patients had between 2 and 4 positive antibodies at their respective maximal follow-up period. The specificity was 100 % for S1+S2, S2 and RBD, 98.3% for N and 92.4% (86.0-96.5%) for S1-directed IgG. The combined use of antigens increases the early sensitivity whilst enforcing high specificity. CONCLUSIONS: Sensitivities at ≥21 days and specificities were excellent, especially for N, S1+S2, S2 and RBD-directed IgG. Caution is however required when interpreting single S1-directed reactivities. Using a multiplex assay complies with the orthogonal testing algorithm of the CDC and allows a better and critical interpretation of the serological status of a patient.

An Outpatient Clinic as a Potential Site of Transmission for an Outbreak of New Delhi Metallo-ß-Lactamase-producing Klebsiella pneumoniae Sequence Type 716: A Study Using Whole-genome Sequencing.

BACKGROUND: The incidence of nosocomial infections due to carbapenem-resistant Klebsiella pneumoniae is increasing worldwide. Whole-genome sequencing (WGS) can help elucidate the transmission route of nosocomial pathogens. METHODS: We combined WGS and epidemiological data to analyze an outbreak of New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae that occurred in 2 Belgian hospitals situated about 50 miles apart. We characterized 74 NDM-producing K. pneumoniae isolates (9 from hospital A, 24 from hospital B, and 41 contemporary isolates from 15 other Belgian hospitals) using pulsed-field gel electrophoresis and WGS. RESULTS: A K. pneumoniae sequence type 716 clone was identified as being responsible for the outbreak with all 9 strains from hospital A and 20 of 24 from hospital B sharing a unique pulsotype and being clustered together at WGS (compared with 1 of 41 isolates from other Belgian hospitals). We identified the outpatient clinic of hospital B as the probable bridging site between the hospitals after combining epidemiological, phylogenetic, and resistome data. We also identified the patient who probably caused the transmission. In fact, all but 1 strain from hospital A carried a Tn1331-like transposon, whereas none of the hospital B isolates did. The patient from hospital A who did not have the Tn1331-like transposon was treated at the outpatient clinic of hospital B on the same day as the first NDM-producing K. pneumoniae-positive patient from hospital B. CONCLUSIONS: The results from our WGS-guided investigation highlight the importance of implementing adequate infection control measures in outpatient settings, especially when healthcare delivery moves from acute care facilities to outpatient clinics.

Host and microbial factors in kidney transplant recipients with Escherichia coli acute pyelonephritis or asymptomatic bacteriuria: a prospective study using whole-genome sequencing.

BACKGROUND: Urinary tract infection is the most common infection among kidney transplant recipients (KTRs). Many transplant physicians fear that host compromise will allow low-virulence strains to cause pyelonephritis in KTRs, so they often treat asymptomatic bacteriuria with antibiotics. Identification of the host/microbe factors that determine the clinical presentation (i.e. pyelonephritis versus asymptomatic bacteriuria) once an Escherichia coli strain enters a KTRs bladder could inform management decisions. METHODS: We prospectively collected all E. coli isolates causing either pyelonephritis or asymptomatic bacteriuria in KTRs at our institution (December 2012-June 2015). Whole-genome sequencing was used to assess bacterial characteristics (carriage of 48 virulence genes and phylogenetic and clonal background). Host parameters were also collected. RESULTS: We analysed 72 bacteriuria episodes in 54 KTRs (53 pyelonephritis, 19 asymptomatic bacteriuria). The pyelonephritis and asymptomatic bacteriuria isolates exhibited a similar total virulence gene count per isolate [median 18 (range 5-33) and 18 (5-30), respectively; P = 0.57] and for individual virulence genes differed significantly only for the prevalence of the pap operon (pyelonephritis 39%,versus asymptomatic bacteriuria 0%; P = 0.002). No other significant between-group differences were apparent for 86 other bacterial and host variables. CONCLUSIONS: Our findings suggest that bacterial adherence plays a role in the pathogenesis of pyelonephritis in KTRs despite significantly altered host urinary tract anatomy and weakened immunity. Whether KTRs might benefit from targeted therapies (e.g. vaccination or inhibitors of fimbrial adhesion) has yet to be studied.

Detection of mecA- and mecC-Positive Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates by the New Xpert MRSA Gen 3 PCR Assay.

An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n = 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n = 160, including 10 mecC-positive MRSA) were accurately categorized.

National surveillance of Staphylococcus epidermidis recovered from bloodstream infections in Belgian hospitals.

OBJECTIVES: The objectives of this study were: (i) to determine the species diversity of CoNS isolated from bloodstream infections collected during a national surveillance study; and (ii) to examine the antimicrobial resistance and genomic diversity among Staphylococcus epidermidis isolates. METHODS: Eighty CoNS were identified by MALDI-TOF. Antimicrobial resistance determination, molecular characterization of resistance and virulence genes, and molecular typing were performed for S. epidermidis isolates. RESULTS: The majority (76%) of CoNS were identified as S. epidermidis. Among these S. epidermidis, 77% were resistant to methicillin [methicillin-resistant S. epidermidis (MRSE)] and showed multiresistance to other antimicrobials. Genes implicated in resistance were erm(C), erm(A) and msr(A) for erythromycin, aacA-aphD and aadC for aminoglycosides, tet(K) for tetracycline and mupA for high-level resistance to mupirocin. Molecular typing showed that 34/40 MRSE isolates (85%) belonged to clonal complex (CC) 2 that could be subdivided into CC2-I (ST2) and CC2-II (ST5, ST59 and ST88). In contrast, methicillin-susceptible S. epidermidis displayed high genomic diversity. The majority (70%) of S. epidermidis isolates contained an icaA or arcA gene. The icaA gene was found in the CC2-I subgroup, whereas arcA was more common in methicillin-susceptible S. epidermidis. CONCLUSIONS: S. epidermidis was frequently recovered among CoNS isolated from bloodstream infections with a high proportion of MRSE being multiresistant. A large number of S. epidermidis belonged to CC2, a clone that is disseminated worldwide. More studies are needed to understand its clonal evolutionary success.

A high-dose aminoglycoside regimen combined with renal replacement therapy for the treatment of MDR pathogens: a proof-of-concept study.

BACKGROUND: Infections caused by MDR Gram-negative (GN) organisms in critically ill patients are a therapeutic challenge. The administration of high-dose aminoglycoside (HDA) therapy coupled with high-flow continuous veno-venous haemodiafiltration (CVVHDF) could allow required high drug peaks to be achieved with acceptable drug elimination. METHODS: All adult patients present on the ICU between October 2009 and July 2014 who had MDR GN sepsis were considered for HDA and high-flow (>45 mL/kg/h) CVVHDF when an isolated pathogen was susceptible or had intermediate susceptibility to aminoglycosides and the patient's condition was not improving with conventional therapy. Optimal antibacterial activity was defined as a peak concentration of at least eight times the MIC. RESULTS: Fifteen patients infected with MDR GN pathogens (11 with Pseudomonas aeruginosa; 10 with abdominal infections and 5 with respiratory infections) were treated with amikacin (n = 11), gentamicin (n = 3) or tobramycin (n = 1) and high-flow CVVHDF. A favourable clinical response was observed in eight (53%) patients, including three in whom microbial eradication was obtained. Six patients were discharged alive from the ICU, and five from the hospital. No renal toxicity was observed among survivors. CONCLUSION: In this cohort of septic patients with MDR GN infections, HDA combined with high-flow CVVHDF represented a valuable therapeutic option. The effectiveness of this approach should be further evaluated in larger studies.

Multicentre evaluation of the Check-Direct CPE® assay for direct screening of carbapenemase-producing Enterobacteriaceae from rectal swabs.

OBJECTIVES: The objective of this study was to evaluate in a multicentre survey the analytical performance of the Check-Direct CPE® assay (CDCPE), a multiplex PCR assay for the detection of carbapenemase-producing Enterobacteriaceae (CPE), directly from rectal swabs. METHODS: Adult patients admitted to a high-risk unit in four participating centres were prospectively screened for CPE carriage by rectal swabbing. Samples were cultured on chromogenic CPE-selective media in the local laboratories. All growing Enterobacteriaceae strains were transferred for confirmation of carbapenemase production by multiplex PCR, together with the faecal swabs for CDCPE, to the coordinating laboratory. RESULTS: Overall, 38 of the 394 samples analysed (9.6%; range 3%-20% per centre) yielded a positive signal for a carbapenemase gene with CDCPE, including 17 samples (4.3%; range 0%-15% per centre) that grew a total of 25 CPE-confirmed isolates (all OXA-48-like producers, including one isolate that simultaneously harboured a VIM-type carbapenemase). No CPE culture-positive samples were missed by CDCPE. Among the 21 samples that were CPE-positive with CDCPE but negative on culture, five were collected from previously known CPE carriers and 6/9 OXA-48-positive signals were detected at one participating centre that was undergoing a hospital-wide outbreak of OXA-48 CPE. When compared with the selective culture, the sensitivity and specificity of CDCPE were 100% and 94%, respectively. CONCLUSIONS: This study showed the value of CDCPE as a tool for screening CPE carriage in an epidemiological setting with a high prevalence of OXA-48 CPE. However, the potential added value for infection control management remains to be demonstrated.

Evaluation of the automated Vitek 2 system for detection of various mechanisms of macrolide and lincosamide resistance in Staphylococcus aureus.

We evaluated the performance of the automated Vitek 2 system against disk diffusion for susceptibility testing of Staphylococcus aureus strains showing various resistance mechanisms to macrolides and lincosamides (ML). The Vitek 2 system showed 100% concordance with the D-zone test in detection of the most common resistance mechanisms to ML, including methylase and efflux systems.

Performance of the Verigene Gram-negative blood culture assay for rapid detection of bacteria and resistance determinants.

Nonduplicate blood cultures that were positive for Gram-negative bacilli (n = 125) were tested by the Verigene Gram-negative blood culture (BC-GN) assay; 117 (90.7%) isolates were members of the panel. For identification and resistance markers, the agreements with routine methods were 97.4% (114/117) and 92.3% (12/13). The BC-GN assay is a rapid and accurate tool for the detection of pathogens from blood cultures and could be integrated alongside conventional systems to enable faster patient management, but the clinical benefits should be further evaluated.

Gonococcal aneurysm of the ascending aorta: case report and review of Neisseria gonorrhoeae endovascular infections.

We present the case of a man with a bicuspid aortic valve who presented with persistent fever. Blood cultures yielded Neisseria gonorrhoeae, and the diagnosis of infected mycotic aneurysm was confirmed by detection of the bacterial genome in the aortic wall. The patient was cured with surgery and intravenous ceftriaxone.

Case-control study of drug monitoring of ß-lactams in obese critically ill patients.

Severe sepsis and septic shock can alter the pharmacokinetics of broad-spectrum ß-lactams (meropenem, ceftazidime/cefepime, and piperacillin-tazobactam), resulting in inappropriate serum concentrations. Obesity may further modify the pharmacokinetics of these agents. We reviewed our data on critically ill obese patients (body mass index of ≥ 30 kg/m(2)) treated with a broad-spectrum ß-lactam in whom therapeutic drug monitoring was performed and compared the data to those obtained in critically nonobese patients (body mass index of <25 kg/m(2)) to assess whether there were differences in reaching optimal drug concentrations for the treatment of nosocomial infections. Sixty-eight serum levels were obtained from 49 obese patients. There was considerable variability in ß-lactam serum concentrations (coefficient of variation of 50% to 92% for the three drugs). Standard drug regimens of ß-lactams resulted in insufficient serum concentrations in 32% of the patients and overdosed concentrations in 25%. Continuous renal replacement therapy was identified by multivariable analysis as a risk factor for overdosage and a protective factor for insufficient ß-lactam serum concentrations. The serum drug levels from the obese cohort were well matched for age, gender, renal function, and sequential organ failure assessment (SOFA) score to 68 serum levels measured in 59 nonobese patients. The only difference observed between the two cohorts was in the subgroup of patients treated with meropenem and who were not receiving continuous renal replacement therapy: serum concentrations were lower in the obese cohort. No differences were observed in pharmacokinetic variables between the two groups. Routine therapeutic drug monitoring of ß-lactams should be continued in obese critically ill patients.

Verocytotoxin-producing Escherichia coli O128ab:H2 bacteremia in a 27-year-old male with hemolytic-uremic syndrome.

Verocytotoxin-producing Escherichia coli (VTEC) strains of serotype O128ab:H2 were isolated from blood and stool of a 27-year-old male presenting diarrhea-associated hemolytic-uremic syndrome complicated by bacteremia. This report once again illustrates the pathogenic potential of a non-O157 VTEC strain carrying a virulence profile previously associated with mild disease.

Long-Term Dynamic of Anti-TrimericS and Anti-RBD Antibodies in Naive and COVID-19 Recovered mRNA-1273 Vaccine Recipients.

OBJECTIVE: Patients and physicians are increasingly requesting their clinical laboratory to provide SARS-CoV-2 serology interpretation. Our study aimed to assess the evolution of SARS-CoV-2 antibodies in Moderna-vaccinated health care workers. METHODS: We analyzed the evolution of mRNA-1273 (Moderna)-elicited antibodies by 2 high-throughput assays, TrimericS IgG (Diasorin) and SARS-CoV-2 IgG-II (Abbott). RESULTS: After the first injection, the COVID-19-recovered vaccinees showed a serological response as strong as that observed 1 month after the second injection in participants without COVID-19 history. Although remaining above the positivity thresholds, the TrimericS immunoglobulin G (IgG) and anti-RBD (receptor-binding domain) IgG levels fell considerably between 1 and 7 months postvaccination, dropping to 10.6% and 13% for the COVID-19 recovered subgroup and to 11.7% and 9.3% for the COVID-19 naive subgroup. CONCLUSION: Regardless of the test used, a decrease in circulating anti-SARS-CoV-2 IgG levels should be expected a few months after vaccination. As this decline does not preclude the efficacy of immune response, caution is necessary when interpretating postvaccination serological data.

Optimal meropenem concentrations to treat multidrug-resistant Pseudomonas aeruginosa septic shock.

A patient with septic shock due to extensively drug resistant (XDR) Pseudomonas aeruginosa was cured by optimizing the meropenem (MEM) regimen to obtain at least 40% of the time between two administrations in which drug levels were four times higher than the MIC of the pathogen. As the standard drug dose did not achieve these optimal concentrations, the MEM regimen was progressively increased up to 12 g/day (3 g every 6 h in a 3-h extended infusion), which eventually resulted in sepsis resolution. High MEM dosage may represent a valuable therapeutic option for infection due to multidrug-resistant (MDR) strains, and drug monitoring would allow rapid regimen adjustment in clinical practice.

Evaluation of Clearview Exact PBP2a, a new immunochromatographic assay, for detection of low-level methicillin-resistant Staphylococcus aureus (LL-MRSA).

We evaluated the performance of a new immunochromatographic assay (ICA), the Clearview Exact PBP2a, for rapid detection of penicillin-binding protein 2a (PBP2a) in a challenge set of Staphylococcus aureus strains showing MICs to oxacillin of ≤16 mg/liter. The sensitivity and specificity of the ICA were 96.6% and 100%, respectively.

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.

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin.

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.