Routine Diagnostic Microbiology Laboratories Research Articles

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC β-lactamases: is routine detection possible?

Phenotypic characterization of the prevalent AmpC β-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. The prevalence of AmpC β-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Candida auris Identification and Profiling by MALDI-ToF Mass Spectrometry.

MALDI-ToF MS has become the standard method for routine identification of most medically important yeasts in clinical and public health laboratories and has largely replaced phenotypic identification methods as a first-line identification tool. Fungal identification is based on extensive and well-curated mass spectra libraries usually provided by the manufacturer of the MALDI-ToF MS platform; however, many centers do create specialized or in-house database collections to aid analysis. Most MALDI-ToF MS systems offer simple and standardized workflows for the identification of clinically relevant yeasts to species level with a high throughput, high accuracy, and a low overall cost per test. Thismakes MALDI-ToF MS an ideal platformfor use in routine clinical, diagnostic, and research microbiology laboratories which may lack experience or expertise in the identification of pathogenic fungi.In this chapter we review three standard protocols for the proteomic-based identification of Candida auris isolated from cultures of clinical or environmental surveillance samples in diagnostic and research laboratories.

Rapid identification of clinical isolates of Klebsiella pneumoniae using MALDI-TOF MS from North India

Infections caused by multi-drug resistant (MDR) Klebsiella pneumoniae are increasing day by day. K. pneumoniae poses a severe public health concern and causes wide array of healthcare associated infections with limited treatment options. The quick detection of these isolates is of prime importance for the adoption of proper antibiotic treatment and control measures. Also the mis- identification using standard lab-based methods is quite common and hence, the clinical significance of K. pneumoniae complex members is inaccurately defined. Here, authors evaluated the potential of MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight) MS (Mass Spectrometry) to discriminate and quick identification K. pneumoniae complex members. Thus, this study aimed at full MALDI-based approach to rapidly detect the K. pneumoniae isolates up to species level. We also explored the antibiotic resistance profile of K. pneumonia isolates using CLSI (Clinical and Laboratory Standards Institute) guidelines. These results demonstrate the potential of MALDI-TOF MS for precise identification of K. pneumoniae complex members and could be effectively utilized in the routine diagnostic microbiology laboratories.

Is There a Need to Revise the Antibiotic Concentration in Clinical and Laboratory Standards Institute-Recommended Oxacillin Screen Agar?

Introduction: In routine diagnostic microbiology laboratories, Clinical and Laboratory Standards Institute (CLSI) recommends the use of cefoxitin disc, in addition to oxacillin screen agar (OSA) of 6 μg/ml for the detection of methicillin-resistant Staphylococcus aureus (MRSA), whereas minimum inhibitory concentration values of oxacillin for S. aureus are ≤2 μg/ml (susceptible) and ≥4 μg/ml (resistant). Hence, the study was carried out to evaluate the ability of screen agar with lower concentrations of oxacillin to identify the isolates of MRSA and to compare this with cefoxitin disc diffusion (CDD). Materials andMethods: Six hundred and seventy-six isolates of S. aureus were screened for methicillin resistance by OSA with 2 μg/ml and 4 μg/ml and 6 μg/ml of oxacillin concentration as well as CDD. Polymerase chain reaction for mecA gene was carried out for all isolates which grew on OSA 2, 4 and 6 μg/ml regardless of their cefoxitin susceptibility. Latex agglutination test for penicillin-binding protein 2a was performed for the isolates which grew on OSA 2 and or 4 μg/ml but not on OSA 6 μg/ml. Results: Eight per cent of MRSA isolates was missed by using OSA 6 μg/ml, when compared with other methods. Sensitivities of OSA 2 μg/ml, OSA 6 μg/ml and CDD were found to be 100%, 92.5% and 97.5%, respectively, and specificities for the same were found to be 100%, 100% and 98%, respectively. As per FDA criteria, categorical agreement for OSA 2 μg/ml was found to be 100% in comparison with the reference broth microdilution method. No major and very major discrepancies were documented. Conclusion: Similar findings on a larger and more heterogeneous collection of isolates may indicate the need to revise the concentration of OSA to 2 μg/ml for the detection of MRSA.

Impact of MALDI-TOF in the Routine Diagnostic Microbiology Laboratory in Alexandria University, Egypt

Background: In the last few years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) has emerged as a useful tool for identification of microorganisms. Identification of the various bacterial genera has been found to be challenging using the routine phenotypic tests or commercially available automated systems relying on the biochemical identification of bacteria. Objectives: In this study, we aimed to evaluate the possible role of the MALDI-TOF system with its Biotyper software as an accurate and rapid method of bacterial identification. Methodology: This study was conducted over a period of one month, where 299 isolates were retrieved from 523 clinical samples. Results: MALDI TOF identified 275 (92%) isolates to the species level, 287 isolates (96%) to the genus level and no reliable identification for 12 (4%) isolates using the direct formic acid application method. When protein extraction was applied, valid results for species and genus identification were obtained for 296 and 299 isolates respectively, so the overall rate of identification rose to 99% and 100% respectively. Discordant results between MALDI TOF and conventional methods were confirmed by Vitek 2 and 16s rRNA sequencing. Conclusions: MALDI-TOF is a rapid, accurate and inexpensive method for microbial identification that rivals other identification systems available. Its relative ease of use and rapid turnaround time makes it a powerful tool in the microbiology laboratory.

Reads2Type: a web application for rapid microbial taxonomy identification.

BackgroundIdentification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data.ResultsRaw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale.ConclusionsIn comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

Melioidosis: a review

The disease melioidosis, caused by the bacterium Burkholderia pseudomallei, remains an important and sometimes neglected cause of disease in tropical regions of Australia. Infection may present in myriad ways, and diagnosis often requires consideration of this organism prior to culture. Laboratory identification of B. pseudomallei requires specialised testing beyond that available in many routine diagnostic microbiology laboratories. For this reason, cases outside of the traditional endemic zone, often occurring years after initial exposure to the organism, may remain undiagnosed or are delayed in diagnosis. Furthermore, the high levels of intrinsic antimicrobial resistance associated with B. pseudomallei often render empirical therapies ineffective. Health professionals, particularly those in rural and remote areas of Australia, must consider melioidosis in their differential diagnoses and remain abreast of advances in the field of this important emerging disease.

Outbreak Investigation Using High-Throughput Genome Sequencing within a Diagnostic Microbiology Laboratory

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla(CTX-M-15) extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications.

Differentiation of Streptococcus pneumoniae from Nonpneumococcal Streptococci of the Streptococcus mitis Group by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

The differentiation of species within the Streptococcus mitis group has posed a problem in the routine diagnostic microbiology laboratory for some time. It also constitutes a major weakness of recently introduced matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) fingerprinting systems. As the phylogenetic resolution of the spectral similarity measures employed by these systems is insufficient to reliably distinguish between the most closely related members of the group, the major pathogen Streptococcus pneumoniae is frequently misidentified. In this study, a comparative analysis of MALDI-TOF spectra of several species from the S. mitis group has been performed in order to identify single peaks that could be used to improve mass spectrometry-based identification of the respective species. A characteristic peak profile could be identified that unambiguously distinguished the 14 S. pneumoniae isolates studied from 33 nonpneumococcal isolates of the S. mitis group. In addition, specific peak combinations could be assigned to other members of the group. The findings of this study suggest that it is possible to distinguish different species of the S. mitis group by close analysis of their mass peak profiles.

Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications

ABSTRACTIdentification of microbial pathogens still relies primarily on culture and phenotypic methods, which is labour-intensive and time-consuming. In this study, identification of bacteria with valid standard identification using BD Phoenix, API panels and other recommended procedures is compared to identification with matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry using the MALDI Biotyper (Bruker Daltonics) in the setting of a routine NHS diagnostic microbiology laboratory. In total, 928 bacterial isolates obtained from blood (n=463), wounds and pus (n=208), respiratory tract (n = 100), faeces (n=86) and urines (n=71) were analysed. There were 721 (77.7%) isolates with a MALDI Biotyper score ≥2.0, indicating secure genus and probable species identification; and 149 (16.1%) isolates with a score ≥1.7 and <2.0 indicating probable genus identification. The isolates with scores of ≥2.0 and ≥1.7 comprised 31 and 33 genera and 65 and 67 species, respectively. Overall, 99.4% and 99.1% of organism identifications were in agreement between the MALDI Biotyper and conventional identification at the genus level, and 89.3% and 87.8% at species level when analysing organisms with MALDI Biotyper scores ≥2.0 and ≥1.7, respectively. With many but not all organisms, identification at the genus level is sufficient; however, MALDI Biotyper separation of 208 staphylococci into Staphylococcus aureus and coagulase-negative staphylococci was always correct when scores were ≥1.7. First results were obtained after 5–10 min and analysis of a full 96-well target plate was completed in approximately 90 min. Substantial savings of between £1.79 and £2.56 per isolate, depending on the cost model of acquisition of the MALDI Biotyper system and number of isolates tested, would be realised when all 928 isolates were identified using the MALDI Biotyper and disk-susceptibility testing when compared to the cost for 618 Phoenix ID panels and 158 API panels and disk-susceptibility tests only (i.e., not taking into account costs incurred for identification of the remaining 152 mixed isolates). Microbial identification by MALDI Biotyper offers a rare opportunity for significant cost-neutral or even cost-saving quality improvements in medical diagnostics.

Evaluation of the Detect-ReadyTM MRSA assay for use in a routine diagnostic microbiology laboratory: Category: Scientific free paper

Evaluation of the Detect-ReadyTM MRSA assay for use in a routine diagnostic microbiology laboratory: Category: Scientific free paper

Development of a novel assay method for colistin sulphomethate

Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.

Diagnosis of mycobacteria in the routine diagnostic laboratory

Non-tuberculous mycobacteria (NTM) have become increasingly important over the last 20 years. These mycobacteria are classified as ?rapid growers? (growth in 7 days or less) or slow growers (growth of isolated colonies in more than 7 days). The rapidly growing mycobacteria (RGM) are the group of mycobacteria which are being recognised in cultures performed in routine diagnostic microbiology laboratories. The increase in notification rate of RGM is partially due to a heightened alertness for mycobacteria by clinicians and laboratory scientists and technicians. However, diagnostic delays remain relatively common, with mycobacteria being considered only following failed treatment with empiric therapy.

Laboratory detection and investigation of reduced susceptibility to vancomycin in oxacillin-resistant Staphylococcus aureus.

The discovery of strains of Staphylococcus aureus with reduced susceptibility to glycopeptides in Japan in 1997 heralded increasing problems with the treatment and infection control of this organism [1]. Since this first report, similar strains have been discovered worldwide, including in Australia [2]. A major difficulty facing routine diagnostic microbiology laboratories has been choosing an appropriate methodology for the detection of these organisms. The fact that heteroresistance to vancomycin occurs with some strains has confused both the technical aspects and the terminology used to describe them. Routine methods used for determining vancomycin susceptibility in Staphylococcus aureus, such as NCCLS

Rapid identification of fungi by sequencing the ITS1 and ITS2 regions using an automated capillary electrophoresis system.

We developed a standardized DNA sequence-based approach for the accurate and timely identification of medically important fungi by sequencing polymerase chain reaction (PCR) products with a rapid automated capillary electrophoresis system. A simple DNA extraction method and PCR amplification using universal fungal primers was used to amplify ribosomal DNA from a range of clinical isolates and reference strains. The entire internal transcribed spacer (ITS) 1-5.8S-ITS2 ribosomal DNA region was sequenced using automated dye termination sequencing for 89 clinical isolates. These had previously been identified by traditional methods and included 12 ascomycetous yeast species, three basidiomycetous yeast species, eight dermatophyte species and two thermally dimorphic fungi, Scedosporium prolificans and S. apiospermum. Furthermore, 21 reference strains representing 19 different Candida species, Geotrichum candidum and Malassezia furfur were also sequenced as part of this study and were used either as standards for sequence-based comparisons, or as assay controls. Sequence-based identification was compared to traditional identification in a blinded manner. Of the clinical isolates tested, 88/89 had DNA sequences that were highly homologous to those of reference strains accessioned in GenBank, and 87/89 gave a sequence-based identification result that correlated with the traditional identification. In contrast to relatively slow conventional methods of identification, a sequence-based identification from a pure culture can be obtained within 24 h of a DNA extraction carried out after a minimal period of culture growth. We conclude that this approach is rapid, and may be a more accurate cost-effective alternative than most phenotypic methods for identification of many medically important fungi frequently encountered in a routine diagnostic microbiology laboratory.

Haemolysin occurrence amongAeromonas hydrophila,Aeromonas caviae andAeromonas sobria strains isolated from different aquatic ecosystems

A total of 909 Aeromonas spp. isolates from different aquatic ecosystems were tested for haemolysin production by both sheep and horse blood agar-plate assays and by rabbit erythrocytes in broth assay. A comparison of these different methods was undertaken in order to appreciate their capacity to evaluate the haemolytic activity of Aeromonas spp. isolated from aquatic ecosystems. The haemolytic activity was associated particularly with A. hydrophila and A. sobria (about 95% of strains), whereas A. caviae did not produce haemolysin (about 95% of strains). A method suitable for use in routine diagnostic microbiology laboratories is proposed for quantifying both groups of A. hydrophila/A. sobria and A. caviae in environmental water.

遺伝子増幅法（ＰＣＲ）による志賀赤痢菌様毒素遺伝子の検出とその型別法

Fourteen isolates of E. coli O157:H7 and five isolates of S. dysenteriae type-1 were examined by polymerase chain reaction (PCR) for the structural genes (slt-I or slt-II), encoding Shiga-like toxins (SLTs). The two primer pairs (V1; 5'AGTTAATGTGGTGGCGAA and V2; 5'GACTGCGTCAGTGAGGTT for SLT-I, V3; 5'TTCGGTATCCTATTCCCG and V4; 5'TCTCTGGTCATTGTATTA for SLT-II) used were of the same positions representing the DNA sequence covering 471bp of the slt-I or slt-II. A 5-microliter portion of boiled bacterial culture broth was used as template DNA in a PCR-reaction mixture of 50 microliters. Two classes, slt-I alone or both slt-I and slt-II, were recognized in E. coli strains. All of S. dysenteriae type-1 strains examined contained slt-I alone. Our results indicate that PCR using these primer pairs is a simple, rapid, sensitive, and specific method and suitable for use in routine diagnostic microbiology laboratories.

A simplified method for detecting the heat-labile enterotoxin of Escherichia coli.

A tissue-culture method is described that is suitable for screening large numbers of Escherichia coli isolates for heat-labile enterotoxin production. The method uses readily available laboratory media for culture of the E. coli and does not require cell-free extracts. Antibiotics are used to increase toxin production to levels that allow cultures from several different colonies to be pooled for testing, thus minimising the number of tissue-culture monolayers required. Y1 mouse adrenal cells are used in microplate cultures. The method is simple, sensitive and economical and is suitable for use in a routine diagnostic microbiology laboratory.

Laser nephelometric semi-automated system for rapid bacterial susceptibility testing.

Experiments with a semi-automated susceptibility test system are described. The system was based on a laser nephelometer which was used to estimate extinction in broth cultures, and ready-to-use cuvettes containing antibiotics. Each antibiotic was tested at two concentrations, the cuvettes were manually inoculated and the results were obtained after 3-5 h incubation in an agitator-incubator. The system was used to test 70 strains (mainly Gram-negative) for susceptibility to 14 antibiotics. The results were compared with the minimal inhibitory concentration (MIC) determined in a solid medium and partially with the ABAC semi-automatic antibiotic sensitivity test. No major discrepancy was observed between the semi-automated laser nephelometric system and MIC and the results were in complete agreement in 91.2%. The laser nephelometric system was found slightly superior to the ABAC system which showed a full accord rate of 88% with MIC. Since the method used a common apparatus in protein immunochemistry, it was found applicable in routine diagnostic microbiology laboratories.