Isabel Montesinos

Trends in production of extended-spectrum beta-lactamases among Enterobacteriaceae of clinical interest: results of a nationwide survey in Belgian hospitals.

OBJECTIVES to assess the frequency and diversity of extended-spectrum β-lactamases (ESBLs) in Enterobacteriaceae isolates in Belgium. METHODS during 2006 and 2008, non-duplicate clinical isolates of Enterobacteriaceae resistant to ceftazidime and/or cefotaxime were collected in 100 Belgian hospitals. ESBL production was confirmed by phenotypic and genotypic tests. MICs of 13 antimicrobial agents were determined by Etest. ESBL-encoding genes were identified by PCR sequencing and the bla(CTX-M) environment was characterized by PCR mapping. Selected isolates were genotyped by PFGE, multilocus sequence typing analysis and phylogenetic grouping by PCR. RESULTS overall, 733 isolates were confirmed as ESBL producers. Carbapenems and temocillin were active against ≥ 95% of all tested isolates. Co-resistance to co-trimoxazole and to ciprofloxacin was found in almost 70% and 80% of the strains, respectively. Overall, Escherichia coli (49%), Enterobacter aerogenes (32%) and Klebsiella pneumoniae (9%) represented the most prevalent species. Isolates harboured predominantly TEM-24 (30.7%), CTX-M-15 (24.2%) and TEM-52 (12.1%). Compared with 2006, the proportion of CTX-M-type enzymes increased significantly in 2008 (54% versus 23%; P < 10(-6)), mostly linked to a rising proportion of CTX-M-15-producing E. coli. TEM-24 decreased (19% in 2008 versus 43% in 2006; P < 10(-6)) during the same period, while the prevalence of TEM-52 remained unchanged (10% in 2008 versus 14% in 2006; not significant). Over 80% of the CTX-M-15-producing E. coli isolates clustered into a single PFGE type and phylogroup B2, corresponding to the sequence type (ST) 131 clone. Intra- and inter-species gene dissemination (CTX-M-15, CTX-M-2 and CTX-M-9) and wide epidemic spread of the CTX-M-15-producing E. coli ST131 clone in several Belgian hospitals were observed. CONCLUSIONS the rapid emergence of multiresistant CTX-M-15-producing E. coli isolates is of major concern and highlights the need for further surveillance in Belgium.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing KPC-2 carbapenemase in Belgium.

Sir, The Klebsiella pneumoniae carbapenemase (KPC) is a class A b-lactamase that confers resistance to virtually all b-lactams including carbapenems. Initially reported in the USA in a Klebsiella isolate in 1996, KPC-producing K. pneumoniae have been responsible for large nosocomial outbreaks in the USA, Israel and Greece where these isolates are now endemic in hospitals. The rapid dissemination of KPC enzymes among and across different enterobacterial species is likely to be due to the localization of blaKPC genes on transferable broad host range plasmids and their association with a transposon, but is also linked with a disseminated international clone of KPC-producing K. pneumoniae isolates of sequence type (ST) 258. Here we report on the first three cases of KPC-2-producing ST258 K. pneumoniae that were detected in Belgium, subsequent to the transfer of patients from three Greek hospitals. In 2009, a patient with high-grade bladder neoplasm with pelvic metastases was transferred from a hospital in Greece to St-Pierre University Hospital in Brussels. Six weeks later, a patient with severe trauma was transferred from another hospital in the same region of Greece to the intensive care unit (ICU) of the same Brussels hospital. Finally, a few days later, the third polytrauma patient was transferred from the ICU of a hospital in Rhodes to the ICU of Erasme University Hospital in Brussels. Upon admission, a urine culture for the first patient and rectal swabs from the two other patients revealed the presence of multidrug-resistant K. pneumoniae (isolates Hk1, Hk2 and Rh1, respectively) with reduced susceptibility to carbapenems. No secondary local transmission occurred in the two hospitals following the rapid implementation of strict infection control measures. MICs produced using the Vitek2 system (N046 AST cards) and Etest (AB BIODISK, Solna, Sweden) were interpreted according to clinical breakpoints from the European Committee for Antimicrobial Susceptibility Testing (EUCAST). The three multidrug-resistant isolates showed high-level resistance to all penicillins (including temocillin) and to all expanded-spectrum cephalosporins except cefepime to which they all remained borderline susceptible (MICs of 8–16 mg/L) (Table 1). Interestingly, the three isolates appeared to be of intermediate susceptibility to meropenem using the Vitek2 system (MIC of 4–8 mg/L) while they were resistant to meropenem by Etest (MIC of 32 mg/L). All the isolates remained susceptible to gentamicin but were of intermediate

Evaluation of the Bio-Rad Geenius HIV-1/2 test as a confirmatory assay

OBJECTIVES We have evaluated the recently Conformité Européenne (CE)-marked Bio-Rad Geenius human immunodeficiency virus (HIV)1/2 as a rapid and simple alternative to western blot for confirmation of HIV screening results. METHODS A total of 160 serum samples were tested: 44 HIV-1 reactive samples by a fourth-generation Murex HIV Ag/Ab and/or Vidas HIV Duo Ultra, five HIV-2 reactive samples, 15 HIV-1 non-B subtype samples and 11 confirmed HIV-1 early seroconversion samples, 72 nonreactive samples, eight indeterminate samples by MP HIV BLOT 2.2 confirmed negative after follow-up and five low-reactive samples by enzyme immunoassay (EIA) negative by MP HIV BLOT 2.2. The samples were tested according to the manufacturers guidelines. RESULTS The overall sensitivity for Bio-Rad Geenius HIV1/2 assay was 92%. Five out of 11 early seroconversion samples were tested positive, four negative and two indeterminate. All HIV-1 non-B subtype samples were tested positive. Two out of the five HIV-2 reactive samples were tested positive HIV-2, two positive HIV-2 with HIV-1 cross-reaction and one HIV positive untypable. After excluding early seroconversion samples, the sensitivity of Bio-Rad Geenius HIV1/2 assay reached 100%. Overall specificity was 96%. All HIV negative serums by fourth-generation EIA were tested negative. All five low-reactive samples by EIA, negative by HIV BLOT 2.2 were tested negative by Bio-Rad Geenius HIV1/2. Two out of the eight indeterminate samples by MP HIV BLOT 2.2 that were confirmed negative after follow-up were tested indeterminate and one invalid, the other five were negative. After excluding these last 13 samples, the specificity of Bio-Rad Geenius HIV1/2 assay reached 100%. In comparison with MP HIV BLOT 2.2, the Bio-Rad Geenius HIV1/2 assay was markedly time saving, allowed full traceability, automatic reading and interpretation. CONCLUSIONS The Bio-Rad Geenius HIV1/2 confirmatory system represents a reliable alternative to other confirmatory assays in HIV testing algorithms and provides clear improvement in quality management.

Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital

ABSTRACT Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR34/L98H was the most prevalent mutation (46.7%), followed by TR46/Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

New case of azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation in Belgium

Department of Microbiology, Erasme Hospital, Universite Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium; Department of Microbiology and Immunology, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium; Department of Infectious Diseases, Erasme Hospital, Universite Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium; Department of Pneumology, Erasme Hospital, Universite Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium

Evaluation of the new LIAISON® CMV IgG, IgM and IgG Avidity II assays

BACKGROUND The diagnosis of CMV infection is challenging and the quality of serological laboratory testing is critical, especially in pregnancy and in the determination of transplant recipients and donors serostatus. OBJECTIVES Evaluate the performances of the new LIAISON(®) CMV II line: LIAISON(®) CMV IgG II, LIAISON(®) CMV IgM II and LIAISON(®) CMV IgG Avidity II in comparison with the routine methods used in our laboratory. STUDY DESIGN The evaluation of LIAISON(®) CMV IgG II and LIAISON(®) CMV IgM II was performed on both prospective routine samples and retrospective selected samples for a total of 383 sera. CMV IgG avidity was assessed with 88 samples. RESULTS The overall agreement was 98.8% for the IgG and 95% for the IgM on the routine population. On selected retrospective samples, excellent agreement was found in the seronegative and past infection groups. In the recent infection group, discordances were observed in 7.1% of IgG and 13.1% of IgM. No recent infection was missed with LIAISON(®). Avidity agreement with VIDAS(®) was 81%. On 51 sera with a known time of infection, no high avidity was found in the group infected for less than 3 months and 82% of the samples showed a high avidity in the group infected for more than 3 months. CONCLUSION The performances of the fully automated LIAISON(®) CMV II line assays are comparable to those of the reference methods used in our lab for both prospective and selected populations. This new CMV line is a useful tool for the diagnosis of CMV infections and CMV immune status in clinical settings.

Clinical evaluation of a multi-parameter customized respiratory TaqMan(®) array card compared to conventional methods in immunocompromised patients.

Abstract Background Respiratory viral infections can cause significant morbidity and mortality in immunocompromised patients. Conventional tests routinely available at most institutions are limited by the number of detectable pathogens, by a poor sensitivity and/or a long turnaround time. Objectives To compare the performance of routine conventional testing with direct fluorescent antibody assays and viral culture to a customized TaqMan® array card (TAC) real-time PCR method, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Study design We collected 143 respiratory samples from 120 symptomatic immunocompromised patients. Samples for which conventional and TAC results were discordant underwent further verification testing. Results The TAC assay identified viral pathogens in more samples than did conventional testing (77/143 versus 27/143; McNemar P <0.0001), even when TAC results for viruses that could not be detected by conventional testing were excluded from analysis (59/143 versus 26/143; P <0.0001). In addition, the TAC assay identified 18 samples with non-viral pathogens. Verification testing confirmed positive TAC results for 50 out of 55 samples for which conventional testing was negative. Two out of three samples with a positive conventional test but negative TAC result were confirmed positive. A viral and a total pathogen co-infection rate of 5.6% and 11.8% were found, respectively. Conclusions The customized TAC assay resulted in a significantly increased identification of respiratory viruses. This study provides a practical real-life assessment of the performance of the TAC assay in a population for whom rapid and accurate diagnosis of viral and atypical pathogens is crucial for appropriate clinical management.

HCV false positive immunoassays in patients with LVAD: A potential trap!

BACKGROUND Left ventricular assist devices (LVAD) are a therapeutic choice for patients with advanced heart failure prior cardiac transplantation. Patients with a LVAD implant are frequently monitored for hepatitis C virus (HCV) as a positive result may be an exclusion criterion for transplantation. OBJECTIVES To determine the rate of false positive results with immunoassays for HCV antibodies in a LVAD population. STUDY DESIGN Between June 2011 and January 2015, HCV antibody testing using a chemiluminescent immunoassay (CLIA) (Liaison, Diasorin) was performed for 32 patients prior and post LVAD implantation. A HCV reactive result by CLIA was repeated and further tested by an enzyme linked fluorescent assay (ELFA) (VIDAS, bioMérieux). For patients with a positive HCV CLIA and ELFA test, immunoblot and HCV RNA detection were performed. RESULTS Prior to LVAD implantation, all patients showed a negative HCV serology. After LVAD implantation, 19 patients (59%) had positive results for HCV antibody using CLIA and ELFA technologies. The HCV immunoblot was negative for 17 patients and indeterminate for two patients. For 15 patients, HCV RNA detection was performed and was undetectable. Actually, no HCV infections were observed among those who were tested for HCV RNA. CONCLUSIONS HCV serological tests routinely used in our laboratories are not reliable in patients with cardiac devices. A positive CLIA and/or ELFA reaction in patients with a LVAD should be confirmed by HCV immunoblot and by HCV RNA PCR detection in order to rule out a HCV infection.

When you can't see the wood for the trees. Mucor circinelloides: A rare case of primary cutaneous zygomycosis.

A patient with refractory diffuse lymphoma treated for pulmonary invasive aspergillosis developed a concomitant primary cutaneous mucormycosis. The mucormycete was identified by sequencing as Mucor circinelloides. This case confirms the importance of a rapid pathogen diagnosis in immunocompromised patients and the usefulness of molecular methods for identification of rare fungal species.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients

Abstract Background Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. Objectives First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. Study design We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Results The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p <0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p >0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. Conclusions Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.