Rational Use of Microbiological Tests in the Diagnosis of Central Nervous System Infections Using Restrictive Criteria: a Retrospective Study.

Central nervous infections, mostly represented by meningitis and encephalitis, remain a diagnostic challenge despite substantial advances in microbiological tools in recent years. Meanwhile, extensive microbiological workups, which often prove to be irrelevant retrospectively, continue to be processed on a large scale, therefore leading to unnecessary costs. The main goal of this study was to evaluate a systematic approach enabling more rational use of microbiological tools in the setting of community-acquired central nervous system infection diagnosis. In this single-center descriptive study, the modified Reller criteria were retrospectively extended to all neuropathogens tested in cerebrospinal fluid (CSF) samples with the FilmArray meningitis/encephalitis panel (BioFire Diagnostics, LLC) and bacterial culture. The inclusion period was 30 months. In total, 1,714 fluid (CSF) samples analyzed from 1,665 patients over 2 and a half years were reported. According to the retrospective application of the modified Reller criteria, microbiological testing was considered unnecessary in 544 CSF samples. Fifteen positive microbiological results were found among these samples, interpreted either as inherited chromosomally integrated human herpesvirus 6 (HHV-6), a false-positive result, or a true microbial detection without clinical relevance. No CNS infection case would have been missed if these analyses were not carried out, while about one-third of all meningitis/encephalitis multiplex PCR panels would have been saved. Our retrospective analysis suggests that the modified Reller criteria could be safely applied to all microbiological tests performed in CSF, thereby saving substantial costs. IMPORTANCE Microbiological testing in general and in the setting of central nervous system (CNS) infection in particular are often excessive, leading to superfluous laboratory work and costs. In this regard, restrictive criteria, named Reller criteria, have been developed to reduce unnecessary CSF herpes simplex virus 1 (HSV-1) PCR testing when suspecting encephalitis. These criteria were then adapted for increased safety to become the modified Reller criteria. This retrospective study aims at evaluating the safety of these criteria when applied to CSF microbiological testing in general, including multiplex PCR, direct examination, and bacterial culture. The postulate was that a CNS infection can be excluded if none of these criteria is present. According to our data set, no CNS infection would have been missed if the modified Reller criteria would have been applied to save microbiological tests. This study therefore proposes a simple way to reduce unnecessary microbiological testing in the context of CNS infection suspicion.

From genotype to antibiotic susceptibility phenotype in the order Enterobacterales: a clinical perspective.

OBJECTIVES: Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in the order Enterobacterales. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacterales species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. METHODS: We sequenced (Illumina MiSeq, 2×250 bp) 187 clinical isolates from species possessing (n = 98) or not (n = 89) an intrinsic AmpC-type cephalosporinase. Phenotypic antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. Piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and ciprofloxacin were considered for prediction. RESULTS: A total of 1496 isolate/antibiotic combinations (187 isolates × 8 antibiotics) were considered. In 230 cases (15.4%), no attempt of prediction was made because it could not be supported by current knowledge. Among the 1266 attempts, 1220 (96.4%) were correct (963 for predicting susceptibility and 257 for predicting resistance), 24 (1.9%) were major errors (MEs) and 22 (1.7%) were very major errors (VMEs). Concordance were similar between non-AmpC and AmpC-producing Enterobacterales (754/784 (96.2%) vs 466/482 (96.7%), chi-square test p 0.15), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (19/784 (2.4%) vs 3/466 (0.6%), chi-square test p 0.02). The majority of VMEs were putatively due to the overexpression of chromosomal genes. CONCLUSIONS: In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacterales species. However, more knowledge about the mechanisms underlying the derepression of AmpC are needed.

Comparison of the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for routine antimicrobial disc diffusion susceptibility testing.

OBJECTIVES: This study investigated the agreement at the categorical level between the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for antimicrobial disc diffusion susceptibility testing. METHODS: The 338 clinical strains (67 Pseudomonas aeruginosa, 19 methicillin-resistant Staphylococcus aureus, 75 methicillin-sensitive S. aureus and 177 Enterobacterales isolates) analysed in this study were non-duplicate isolates obtained from consecutive clinical samples referred to the clinical bacteriology laboratory at Geneva University Hospitals between June and August 2019. For the WASPLab the inoculum suspension was prepared in strict accordance with the manufacturer's instruction (Copan WASP srl, Brescia, Italy) by adding 2 mL of the 0.5 McFarland primary suspension used for the SIRscan analysis into a sterile tube filled with 4 mL of sterile saline (1:3 dilution). The inoculum (2 × 30 µL loop/spreader) was spread over the entire surface of Mueller-Hinton agar plates according to the AST streaking pattern defined by Copan. The antibiotic discs were dispensed by the WASP and inoculated media were loaded on conveyors for transfer to the automatic incubators. The plates were incubated for 16 h, and several digital images were acquired. Inhibition zone diameters were automatically read by the WASPLab and were adjusted manually whenever necessary. For the SIRscan 2000 automatic, the antimicrobial disc diffusion susceptibility testing was performed according to the EUCAST guidelines. The gradient strip method was used to resolve discrepancies. RESULTS: The overall categorical agreement between the compared methods reached 99.1% (797/804; 95% CI 98.2%-99.6%), 99.5% (1029/1034; 95% CI 98.9%-99.8%), and 98.8% (2798/2832; 95% CI 98.3%-99.1%) for P. aeruginosa, S. aureus and the Enterobacterales, respectively. CONCLUSIONS: WASPLab incorporating the BioRad expert system provides a fully automated solution for antimicrobial disc diffusion susceptibility testing with equal or better accuracy than other available phenotypic methods.

A molecular based diagnosis of positive blood culture in the context of viral haemorrhagic fever: proof of concept.

OBJECTIVES: The aim of this study was to evaluate the possibility of using a PCR-based panel to identify bacterial and fungal bloodstream infections in the setting of suspected or confirmed viral haemorrhagic fever. METHODS: The accuracy of the FilmArray® Blood Culture Identification Panel (BCID) assay was assessed to identify the common bacterial and fungal pathogens associated with bloodstream infections after positive blood culture inactivation using a guanidinium thiocyanate containing buffer lysis that is commonly used for viral haemorrhagic fever molecular diagnostics. RESULTS: The FilmArray® BCID panel assay detected 95% (19/20) of the pathogens analysed in this study by using both protocols with and without inactivation. Absolute consistency (100%) was observed in all isolates with phenotypes compatible with the presence of the antibiotic resistance genes mecA, vanA, vanB and blaKPC. CONCLUSIONS: The FilmArray® BCID panel assay coupled to inactivation using a guanidinium thiocyanate containing buffer lysis represents a convenient, sensitive and specific diagnostic tool to detect some of the most pathogens associated with bloodstream infections in the context of a suspected or confirmed viral haemorrhagic fever.

Copan WASPLab automation significantly reduces incubation times and allows earlier culture readings.

OBJECTIVE: The aim was to evaluate whether laboratory automation (inoculation and automated incubation combined with timely defined high-resolution digital imaging) may help reduce the time required to obtain reliable culture analysis results. METHODS: We compared the results obtained by WASPLab automation against WASP-based automated inoculation coupled to conventional incubation and manual diagnostic on 1294 clinical samples (483 for the derivation set and 811 for the independent validation set) that included urine, genital tract and non-sterile site specimens, as well as ESwabs for screening of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), extended-spectrum beta-lactamases (ESBLs) and carbapenemase-producing Enterobacteriaceae (CPE). We used sequential routine specimens referred to the bacteriology laboratory at Geneva University Hospitals between October 2018 and March 2019. RESULTS: The detection sensitivity of MRSA and MSSA at 18 hr on WASPLab was 100% (95% confidence interval [CI], 94.48-100.00%). The detection sensitivity of ESBL and CPE at 16 hr on WASPLab was 100% (95% confidence interval [CI], 94.87% to 100.00%). For urine specimens, the similarity was 79% (295/375) between 18 hr and 24 hr of incubation on WASPLab. For genital tract and non-sterile site specimens, the similarity between 16 hr and 28 hr of incubation on WASPLab were 26% (72/281) and 77% (123/159) respectively. Thus, 28 hr was defined as the final incubation time on WASPLab for genital tract and non-sterile site specimens. CONCLUSIONS: The results of this study show that WASPLab automation enables a reduction of the culture reading time for all specimens tested without affecting performances. Implementing the established and duly validated incubation times will allow appropriate laboratory workflows for improved efficiency to be built.

In vivo selection of a multidrug-resistant Aeromonas salmonicida during medicinal leech therapy.

We report the selection in a 15-year-old boy of a multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing Aeromonas salmonicida after medicinal leech therapy that required an antibiotic prophylaxis based on piperacillin/tazobactam and cotrimoxazole. Whole genome sequencing of the strain indeed revealed 13 antibiotic resistance genes, including the ESBL CTX-M-3 and the unusual ß-lactamase SCO-1.

Impact of Exposure of Methicillin-Resistant Staphylococcus aureus to Polyhexanide In Vitro and In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) resistant to decolonization agents such as mupirocin and chlorhexidine increases the need for development of alternative decolonization molecules. The absence of reported severe adverse reactions and bacterial resistance to polyhexanide makes it an excellent choice as a topical antiseptic. In the present study, we evaluated the in vitro and in vivo capacity to generate strains with reduced polyhexanide susceptibility and cross-resistance with chlorhexidine and/or antibiotics currently used in clinic. Here we report the in vitro emergence of reduced susceptibility to polyhexanide by prolonged stepwise exposure to low concentrations in broth culture. Reduced susceptibility to polyhexanide was associated with genomic changes in the mprF and purR genes and with concomitant decreased susceptibility to daptomycin and other cell wall-active antibiotics. However, the in vitro emergence of reduced susceptibility to polyhexanide did not result in cross-resistance to chlorhexidine. During in vivo polyhexanide clinical decolonization treatment, neither reduced polyhexanide susceptibility nor chlorhexidine cross-resistance was observed. Together, these observations suggest that polyhexanide could be used safely for decolonization of carriers of chlorhexidine-resistant S. aureus strains; they also highlight the need for careful use of polyhexanide at low antiseptic concentrations.

Clonal or not clonal? Investigating hospital outbreaks of KPC-producing Klebsiella pneumoniae with whole-genome sequencing.

OBJECTIVES: Whole-genome sequencing (WGS) is a promising tool for identifying transmission pathways in outbreaks caused by multidrug-resistant bacteria. However, it is uncertain how the data produced by WGS can be best integrated into epidemiologic investigations. METHODS: We tested various genomic analyses to identify clonal groups in two distinct outbreaks of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae that occurred in Switzerland in 2013 and 2015. In blinded fashion, we sequenced 12 strains involved in the two outbreaks, respectively, and six that were epidemiologically unrelated. We analysed genomic commonalities from conserved genes to plasmid-borne antibiotic resistance genes (ARGs) and contrasted these results with available epidemiologic evidence. RESULTS: Using WGS, blinded analysts correctly identified the two clusters of strains from the two outbreaks. Nonetheless, the 2015 index strain was found to be slightly different (1-3 single nucleotide variants) from the strains recovered from secondary cases, likely because prior long-term carriage (3 years) by the index patient allowed for genetic mutations over time. Also, we observed occasional loss of ARG-bearing plasmidic fragments in outbreak-causing strains. CONCLUSIONS: Retrospective WGS analysis was successful in identifying clonal groups in both outbreaks. Still, data should be analysed with caution in cases of previous long-term carriage of the studied bacteria.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation.

OBJECTIVE: To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). METHODS: Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. RESULTS: All 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 µg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 µg/mL. CONCLUSIONS: The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Rapid molecular determination of methicillin resistance in staphylococcal bacteraemia improves early targeted antibiotic prescribing: a randomized clinical trial.

Empiric therapy of methicillin-susceptible Staphylococcus aureus (MSSA) infections with vancomycin is associated with poorer outcome than targeted therapy with ß-lactams. Our objective was to evaluate whether rapid determination of methicillin resistance shortens the time from Gram stain to targeted antimicrobial therapy in staphylococcal bacteraemia, thereby reducing vancomycin overuse. This was a single-centre open parallel RCT. Gram-positive cocci in clusters in positive blood culture underwent real-time PCR for rapid species and methicillin resistance determination parallel to conventional microbiology. Patients were randomized 1:1 so that clinicians would be informed of PCR results (intervention group) or not (control group). Eighty-nine patients (intervention 48, control 41) were analysed. MRSA was identified in seven patients, MSSA in 46, and CoNS in 36. PCR results were highly concordant (87/89) with standard microbiology. Median time (hours) from Gram stain to transmission of methicillin-susceptibility was 3.9 (2.8-4.3) vs. 25.4 (24.4-26-7) in intervention vs. control groups (p <0.001). Median time (hours) from Gram stain to targeted treatment was similar for 'all staphylococci' [6 (3.8-10) vs. 8 (1-36) p 0.13] but shorter in the intervention group when considering S. aureus only [5 (3-7) vs. 25.5 (3.8-54) p <0.001]. When standard susceptibility testing was complete, 41/48 (85.4%) patients in the intervention group were already receiving targeted therapy compared with 23/41 (56.1%) in the control group (p 0.004). There was no significant effect on clinical outcomes. Rapid determination of methicillin resistance in staphylococcal bacteraemia is accurate and reduces significantly the time to targeted antibiotic therapy in the subgroup of S. aureus, thereby avoiding unnecessary exposure to vancomycin.

Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and ß-lactam resistance mechanisms.

The purpose of this study was to analyze the molecular mechanisms of ampicillin-resistant Haemophilus influenzae isolated in Geneva, Switzerland. We investigated the association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (with or without ß-lactamase production) and ß-lactam susceptibility. Another main focus for this study was to compare the accuracy of disk diffusion and Etest methods to detect resistance to ampicillin and amoxicillin/clavulanic acid. The antibiotic susceptibility to ß-lactam antibiotics of 124 H. influenzae isolates was determined by disk diffusion and Etest methods, and interpreted by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Alterations in PBP3 were investigated by sequencing the ftsI gene. Of the 124 clinical isolates analyzed, ampicillin resistance was found in 36% (45 out of 124). The rate of resistance to amoxicillin/clavulanic acid was 9% and 0.8%, using EUCAST and CLSI breakpoints respectively. For the 78 ß-lactamase negative ampicillin-susceptible (BLNAS) isolates for which the Etest method indicated a high degree of susceptibility (MIC ≤ 1 mg/L), the disk diffusion method revealed resistance to ampicillin and amoxicillin/clavulanic acid in 33 cases (42%). Most common amino acid substitutions were Asn526Lys and Val547Ile, followed by Asp569Ser, Ala502Val, Asp350Asn, Met377Ile, Ile449Val, and Arg517His. The patterns observed were classified into six groups (IIa, IIb, IIc, IId, III-like, and miscellaneous). Continued characterization of both invasive and respiratory H. influenzae isolates is necessary in order to observe changes in the microbiology and epidemiology of this pathogen that could lead to clinical failure when treated by empirical antibiotic therapy.

[Diagnosis of bacterial gastroenteritis]. / Diagnostic de la gastroentérite bactérienne.

The etiologic agents of acute gastroenteritis are diverse. The diagnosis of bacterial pathogens is particularly challenging given the large amount of vastly diverse indigenous gastrointestinal organisms present in stool. Multiple methods must be used by the clinical microbiology laboratories to diagnose the cause of acute gastroenteritis, including bacterial cultures, ELISA, and microscopy. Due to the limitations of conventional methods, there is still room for improvement in the detection of pathogens by using the molecular methods. This paper discusses these different diagnostic approaches and limitations.

[Rapid tests during respiratory infection: an aid to non-prescription of antibiotics? ]. / Tests rapides lors d'infection respiratoire: une aide à la non-prescription d'antibiotiques?

Due to overuse, we are about to reach the end of the antibiotic era. Each of us is responsible to limit their usage to a minimum. Respiratory infections are the first cause of antibiotic prescriptions. The use of new simple tests available at the bedside can be very useful in this context. The development of sophisticated molecular diagnostic tools, such as "multiorganism" panels, may revolutionize our approach to respiratory infections. The key will be to interpret the results correctly, with due consideration of the statement, "Treat patients, not lab results".

[ESBL and carbapenemases in Enterobacteriaceae]. / Bêtalactamases à spectre étendu et carbapénémases chez les Enterobacteriaceae.

Carbapenemase-producing enterobacteria (CPE) spread all over the world during the last years, causing serious infections with increasing frequency. Very few new drugs active against CPE are expected to be clinically available. Studies summarized in this review show that there is yet room to improve our therapeutic approaches, in the treatment of infections due to CPE.

[Which benefits for the clinician to implement MALDI-TOF/MS in the bacteriology laboratory?]. / Quels bénéfices pour les cliniciens de la mise en place du MALDI-TOF/MS dans te taboratoire de bactériologie?

Mass spectrometry (MALDI-TOF/MS) is a recent technology especially adapted to identify microbial pathogens. It has rapidly established itself as a must for most medical bacteriology laboratories. Its ease of use and speed of execution typically permit providing pathogen identification one day earlier to the clinicians. MALDI-TOF/MS facilitates identification of filamentous fungi and mycobacteria that require particular lab expertise when using conventional methods. This paper highlights the multiple advantages that MALDI-TOF/MS can bring to the physicians in their practice.

Prosthetic reconstruction after surgical resection of fibrous dysplasia of the maxillary and palatine bone.

Fibrous dysplasia is a rare disorder of the bone. It is seen in two main forms of presentation: monostotic and the polyostotic. A case of monostotic fibrous dysplasia of the maxillary and palatine bones in a 22-year old man who received prosthetic reconstruction is presented with a review of the literature.

Prosthetic reconstruction after surgical resection of fibrous dysplasia of the maxillary and palatine bone / Reconstrucción prostética tras la resección quirúrgica de la displasia fibrosa del hueso maxilar y el palatino

Fibrous dysplasia is a rare disorder of the bone. It is seen in two main forms ofpresentation: monostotic and the polyostotic. A case of monostotic fibrous dysplasia of the maxillary and palatine bones in a 22-year old man who received prosthetic reconstruction is presented with a review of the literature.

La displasia fibrosa es un trastorno raro del hueso. Se le ve en dos formas principales: la monostótica y la poliostótica. Junto con la correspondiente revisión de la literatura, se presenta un caso de displasia fibrosa monostótica de los huesos maxilar y palatino en un hombre de 22 anos que recibió una reconstrucción prostética.

[Epidemiology and clinical implications of carbapenemase-producing bacteria in Switzerland]. / Carbapénémases: implications cliniques et épidémiologiques pour la Suisse.

The emergence and global dissemination of carbapenemases represents a major threat to public health. Switzerland has not been spared; we report a case-series of four patients hospitalised in our institution colonised with carbapenemase-producing bacteria. Infections caused by carbapenemase-producing Enterobacteriaceae limit therapeutic options and increase mortality. Detection of carbapenemases is also a challenge for laboratories. It is imperative to implement stringent infection control measures in order to prevent epidemics at the hospital level.

Retropharyngeal abscess in adults.

OBJECTIVES: Retropharyngeal abscess in adults can be life-threatening. The otolaryngologist is on the front line in making the diagnosis and treatment of this disease. The aim of this study is to review the clinical features, the diagnostic tools and the management of retropharyngeal abscesses in adults. PATIENTS AND METHODS: Retrospective study of retropharyngeal abscesses in adults admitted in the ENT department from 2005 to 2010. RESULTS: In total 4 patients were included in this study: mean age of 53 years (range 45 to 62 years), sex ration F/M = 3. Cultures obtained from the abscesses identified group A beta-hemolytic streptococci susceptible to amoxicilline-clavulanate in three cases. The treatment consisted in surgical drainage of the collection and intravenous antibiotics. CONCLUSIONS: Retropharyngeal abscesses in adults are critical infections requiring prompt diagnosis and treatment. Computed tomography scan was the crucial tool for the diagnosis, notably to differentiate cellulitis from abscesses. The management includes intravenous broad-spectrum antibiotics associated, if necessary, with surgical drainage in cases of persistent abscess. The outcome is usually good.

Application and use of various mass spectrometry methods in clinical microbiology.

When confronted with a septic patient or dealing with an emerging epidemic, clinicians, infection control specialists and microbiologists have often felt an immense 'need for speed' while waiting for culture results. Various mass spectrometry (MS) applications are about to answer most of their demands. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS of whole bacterial cells has already greatly shortened the time needed for identification of a positive culture in major diagnostic laboratories in Europe. MS is described in this article, with a special emphasis on the different systems currently commercially available for routine identification. MALDI-TOF MS remains, however, limited by the previous time-consuming culture steps, and is not suited for strain typing in epidemic contexts. These limitations can be overcome by other applications of MS in microbiology. MALDI-resequencing is a rapid method for genotyping, offering comparable results to multilocus sequence typing. New systems of broad-range PCR, associated with analyses of amplicons by electrospray ionization MS, might allow nearly full automation for the direct identification of pathogens in blood, thus bypassing the culture stage. This article describes various applications of MS methods in clinical microbiology, and provides a comparative table of these technologies.