Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

A Study on the Bacteriological Profile of Ascitic Fluids and Their Antibiotic Susceptibility Pattern in a Tertiary Care Hospital.

Background Ascitic fluid culture remains an essential step in the management of all patients with ascites, regardless of their presenting complaints. Diagnostic paracentesis should not be delayed or prevent timely administration of antibiotics, particularly in unstable patients. Hence, it is an essential part of the surveillance system of every hospital to perform ascitic fluid culture and assess the antibiotic susceptibility patterns of bacterial isolates. In view of this perspective, the present study was conducted at Chengalpattu Medical College Hospital, Tamil Nadu, India. Objective The aim of the study is to determine the bacterial isolates of ascitic fluid samples and study their antibiotic susceptibility patterns. Materials and methods Ascitic fluids received in the central laboratory at the Department of Microbiology from various departments were included in this study. Preliminary identification of isolates was performed by direct Gram staining, acid-fast staining, and motility testing by the hanging drop method. Within one hour of receiving the samples, they were plated onto blood agar and MacConkey agar media and incubated for 18-24 hours at 37°C for isolation. Growth was checked, and species identification was done based on conventional methods. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Results In this study, a total of 100 ascitic fluid samples were collected, of which only eight (8%) showed growth. Among the eight isolates, six (75%) were Gram-negative bacilli (GNB). Four (66.66%) of the six GNB were Klebsiella spp., while the remaining two (33.33%) were Escherichia coli. Both Gram-positive cocci were Staphylococcus aureus. All the GNB isolates were susceptible to meropenem, piperacillin-tazobactam, and ceftriaxone, with varying susceptibilities to other drugs. Both Gram-positive isolates were found to be methicillin-sensitive Staphylococcus aureus. Conclusion GNB were the predominant organisms in cases of ascitic fluid infection, and they showed 100% susceptibility to carbapenem drugs (especially meropenem), piperacillin-tazobactam, and ceftriaxone. All these drugs can be kept in reserve for serious infections. Amikacin and gentamicin showed promising susceptibility. These drugs can be started empirically with patients on admission before performing culture. Drug adjustments may be later made based on culture reports.

Helicobacter pylori Antibiotic Resistance: Molecular Basis and Diagnostic Methods.

Helicobacter pylori is one of the most common cause of human infections. Infected patients develop chronic active gastritis in all cases, which can lead to peptic ulcer, atrophic gastritis, gastric cancer and gastric MALT-lymphoma. The prevalence of H. pylori infection in the population has regional characteristics and can reach 80%. Constantly increasing antibiotic resistance of H. pylori is a major cause of treatment failure and a major problem. According to the VI Maastricht Consensus, two main strategies for choosing eradication therapy are recommended: individualized based on evaluating sensitivity to antibacterial drugs (phenotypic or molecular genetic method) prior to their appointment, and empirical, which takes into account data on local H. pylori resistance to clarithromycin and monitoring effectiveness schemes in the region. Therefore, the determination of H. pylori resistance to antibiotics, especially clarithromycin, prior to choosing therapeutic strategy is extremely important for the implementation of these treatment regimens.

Evaluation of phenotypic methods for detection of polymyxin B-resistant bacteria.

Determination of sensitivity to polymyxins has always been a challenge, especially in clinical laboratory routines. This study evaluated two rapid, simple, and inexpensive phenotypic methods to test polymyxin B (PMB) susceptibility in Enterobacterales and non-fermenting Gram-negative bacilli. One hundred isolates were used in the tests. The isolates were collected in three hospitals in southern and southeastern Brazil from 1995 to 2019. We compared broth microdilution (reference method) with the broth disk elution test and modified drop test, using polymyxin B -disk or PMB -powder in 2 concentrations (12 and 16 µg/ml). For the broth disk elution and modified drop test with the concentration of 12 µg/ml, categorical agreement values exceeded 90%. The modified drop test with a concentration of 12 µg/ml and broth disk elution may be excellent for initial screening of polymyxin-resistance in laboratory routines. Moreover, these methods are simple and use inexpensive supplies, and may optimize therapeutic decisions.

Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review.

Staphylococcus aureus is an opportunistic pathogen responsible for a wide range of infections in humans, such as skin and soft tissue infections, pneumonia, food poisoning or sepsis. Historically, S. aureus was able to rapidly adapt to anti-staphylococcal antibiotics and become resistant to several classes of antibiotics. Today, methicillin-resistant S. aureus (MRSA) is a multidrug-resistant pathogen and is one of the most common bacteria responsible for hospital-acquired infections and outbreaks, in community settings as well. The rapid and accurate diagnosis of antimicrobial resistance in S. aureus is crucial to the early initiation of directed antibiotic therapy and to improve clinical outcomes for patients. In this narrative review, I provide an overview of recent phenotypic and molecular diagnostic methods for antimicrobial resistance detection in S. aureus, with a particular focus on MRSA detection. I consider methods for resistance detection in both clinical samples and isolated S. aureus cultures, along with a brief discussion of the advantages and the challenges of implementing such methods in routine diagnostics.

The Role of Cartridge-Based Nucleic Acid Amplification Test (CBNAAT), Line Probe Assay (LPA), Liquid Culture, Acid-Fast Bacilli (AFB) Smear and Histopathology in the Diagnosis of Osteoarticular Tuberculosis.

BACKGROUND: There is a paucity of data on the role of molecular methods in the diagnosis of osteoarticular tuberculosis. The present study was conducted to define the role of molecular (CBNAAT, LPA), phenotypic (AFB smear and culture) and histopathological evaluation in the diagnosis of osteoarticular TB. METHODS: Seventy-seven consecutive cases of osteoarticular tuberculosis were grouped into presumptive TB cases (group A) and presumptive drug-resistant cases (group B). Tissue samples obtained were submitted for CBNAAT, LPA, AFB smear, liquid culture and histological examinations. The diagnostic accuracy of each test was reported against histologically diagnosed cases and in all tests in tandem. RESULTS: Group A and group B had 65 and 12 cases, respectively. The diagnostic accuracy for tuberculosis was 84.62% by CBNAAT, 70.77% by LPA, 86.15% by molecular tests (combined), 47.69% by AFB smear, 50.77% by liquid culture and 87.69% by histology in group A, and 91.67% for CBNAAT, 83.33% for LPA, 91.67% for molecular tests (combined), 25% for AFB smear, 16.67% for liquid culture and 83.33% for histology in group B. The drug resistance detection rate was 4.62% on CBNAAT, 3.08% on LPA, 6.15% on molecular tests (combined) and 1.54% on DST in group A, while it was 33.33% on CBNAAT, 58.33% on LPA, 58.33% on molecular tests (combined) and 16.67% on DST among group B cases. Similar sensitivity rates for the various tests were obtained among both the groups on comparison with histology (taken as denominator). The addition of molecular methods increased the overall diagnostic accuracy (all tests in tandem) from 93.8 to 100% in group A and from 83.3 to 100% in group B cases. CONCLUSION: No single tests could diagnose tuberculosis in all cases; hence, samples should be evaluated by molecular tests (CBNAAT and LPA), AFB smear, culture and histological examinations simultaneously. The molecular tests have better demonstration of drug resistance from mycobacterial culture. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43465-020-00326-w.

Comparative Evaluation of the Modified Carbapenem Inactivation Method for Phenotypic Detection of Guiana Extended-Spectrum ß-Lactamase-Type Carbapenemases in Enterobacterales.

OBJECTIVE: We comparatively evaluated the performance of 3 phenotypic tests for the detection of carbapenemase production. MATERIALS AND METHODS: Carbapenemase production was evaluated using the modified Hodge test (MHT), the modified carbapenemase inhibition method (mCIM), and the Rapidec Carba NP test (RCNP). RESULTS: Among the 170 isolates, 79 were CP-CRE and 91 were non-CP-CRE. The CP-CRE isolates produced GES-5 (n = 66), KPC (n = 4), NDM (n = 7), NDM and OXA-48 (n = 1), and VIM (n = 1). For KPC producers, all 3 methods showed a sensitivity of 75%. The sensitivities of MHT, mCIM, and RCNP were 14.3%, 100%, and 71.4%, respectively, for NDM producers, and 1.5%, 12.1%, and 18.2% for GES-5 producers, respectively. CONCLUSION: The performance of the phenotypic tests varied depending on the type of carbapenemase. For intensive infection control, phenotypic and molecular tests are required for the detection of common and rare types of carbapenemases.

Comparison of Phenotypic MRSA Detection Methods with PCR for mecA Gene in the Background of Emergence of Oxacillin-Susceptible MRSA.

Background: Phenotypic methods for detection of methicillin resistance in Staphylococcus aureus (MRSA) can be inaccurate due to heterogeneous expression of resistance and due to environmental factors that influence the expression of resistance. This study aims to compare various phenotypic methods of detection of methicillin resistance with polymerase chain reaction (PCR) for mecA gene and to detect the presence of oxacillin-susceptible MRSA (OS-MRSA). Materials and Methods: A total of 150 S. aureus isolates were tested using cefoxitin disk diffusion, oxacillin salt agar (OSA), latex agglutination test for penicillin binding protein 2a antigen, chromogenic MRSA ID agar, and mecA PCR. Results: Using PCR as the gold standard, 91 (60.66%) of 150 clinical S. aureus strains were identified as MRSA. Three oxacillin-susceptible (minimum inhibitory concentration ≤2 µg/mL) mecA-positive isolates were classified as OS-MRSA. Among the different phenotypic MRSA detection methods studied, latex agglutination had the highest sensitivity and specificity (98.9% and 98.3%), followed by cefoxitin disk diffusion (95.6% and 98.3%), MRSA ID (97.8% and 83.05%), and OSA (86.81% and 94.92%). Conclusion: The sensitivity of cefoxitin disk diffusion method may be reduced in areas with a high prevalence of OS-MRSA where a combination of cefoxitin disk diffusion test with MRSA ID agar or latex agglutination is recommended.

Biomarkers of Infection and Sepsis.

The role of biomarkers for detection of sepsis has come a long way. Molecular biomarkers are taking front stage at present, but machine learning and other computational measures using bigdata sets are promising. Clinical research in sepsis is hampered by lack of specificity of the diagnosis; sepsis is a syndrome with no uniformly agreed definition. This lack of diagnostic precision means there is no gold standard for this diagnosis. The final conclusion is expert opinion, which is not bad but not perfect. Perhaps machine learning will displace expert opinion as the final and most accurate definition for sepsis.

Accurate identification of clinically important Acinetobacter spp.: an update.

Acinetobacter species have emerged as one of the most clinically important pathogens. The phenotypic techniques which are currently available are insufficient in accurately identifying and differentiating the closely related and clinically important Acinetobacter species. Here, we discuss the advantages and limitations of the conventional phenotypic methods, automated identification systems, molecular methods and MALDI-TOF in the precise identification and differentiation of Acinetobacter species. More specifically, several species of this genus are increasingly reported to be of high clinical importance. Molecular characterization such as of bla OXA-51-like PCR together with rpoB sequencing has high discriminatory power over the conventional methods for Acinetobacter species identification, especially within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

Detection of carbapenemase-producing Pseudomonas aeruginosa by phenotypic and genotypic methods in a tertiary care hospital of East India.

BACKGROUND: Carbapenemase-producing Pseudomonas aeruginosa is a serious threat in hospital infection due to its multidrug resistance. AIM: The aim of the study was to determine the frequency of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa and detect the presence of carbapenemase enzymes in carbapenem-resistant P. aeruginosa (CRPA) isolates by phenotypic and genotypic methods. MATERIAL AND METHODS: Double-disk synergy test [DDST] and combined disk synergy test [CDST]) was performed in CRPA isolates and the prevalence of blaKPC, blaNDM-1, blaIMP, blaVIM, blaSIM, blaSPM, blaGIM, and blaOXA-48 was determined. RESULTS: Of 559 isolates included in the study, a total of 102 isolates were resistant to carbapenem that accounted for overall 18.24% (102/559) prevalence. Of these 102 isolates, 89 (87.25%) isolates were positive by DDST and 95 (93.17%) isolates were positive by CDST. Of 102 CRPA isolates, blaVIM was detected in 30 isolates (30/102, 29.1%), followed by blaNDM-1 in 29 (29/102, 28.4%) isolates and blaSIM and blaGIM in 6 isolates each (6/102, 5.8%). A combination of two carbapenemase genes was detected in 12 isolates, with six (6/102, 5.88%) CRPA isolates harboring with both blaVIM and blaNDM-1 genes. Four isolates were found to harbor a combination of three carbapenem-resistant genes. CONCLUSION: A high rate of carbapenemase production was observed in P. aeruginosa. Coproducers of multiple carbapenemases are also a cause of concern. An in-depth understanding of molecular mechanisms of resistance will be helpful in optimizing patient management and hospital infection control.

Prevalence of blaKPC and its occurrence with other beta-lactamases in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. AIM AND OBJECTIVE: This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. MATERIALS AND METHODS: A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. RESULTS: Among the 370 isolates, 41 were resistant to the carbapenem by disc diffusion and minimum inhibitory concentration tests. Screen test using ertapenem and the boronic acid disk was positive in 14 isolates. Only one isolate harbored KPC gene by PCR, and it was co-produced with SHV-12 and CTX-M-15. CONCLUSION: PCR remains the gold standard for detection of KPC compared with any other phenotypic methods. Early detection of these genes helps in initiating proper antibiotic treatment.

Evaluation of a Culture-Dependent Algorithm and a Molecular Algorithm for Identification of Shigella spp., Escherichia coli, and Enteroinvasive E. coli.

Identification of Shigella spp., Escherichia coli, and enteroinvasive E. coli (EIEC) is challenging because of their close relatedness. Distinction is vital, as infections with Shigella spp. are under surveillance of health authorities, in contrast to EIEC infections. In this study, a culture-dependent identification algorithm and a molecular identification algorithm were evaluated. Discrepancies between the two algorithms and original identification were assessed using whole-genome sequencing (WGS). After discrepancy analysis with the molecular algorithm, 100% of the evaluated isolates were identified in concordance with the original identification. However, the resolution for certain serotypes was lower than that of previously described methods and lower than that of the culture-dependent algorithm. Although the resolution of the culture-dependent algorithm is high, 100% of noninvasive E. coli, Shigella sonnei, and Shigella dysenteriae, 93% of Shigella boydii and EIEC, and 85% of Shigella flexneri isolates were identified in concordance with the original identification. Discrepancy analysis using WGS was able to confirm one of the used algorithms in four discrepant results. However, it failed to clarify three other discrepant results, as it added yet another identification. Both proposed algorithms performed well for the identification of Shigella spp. and EIEC isolates and are applicable in low-resource settings, in contrast to previously described methods that require WGS for daily diagnostics. Evaluation of the algorithms showed that both algorithms are capable of identifying Shigella species and EIEC isolates. The molecular algorithm is more applicable in clinical diagnostics for fast and accurate screening, while the culture-dependent algorithm is more suitable for reference laboratories to identify Shigella spp. and EIEC up to the serotype level.

Rapid phenotypic methods to improve the diagnosis of bacterial bloodstream infections: meeting the challenge to reduce the time to result.

BACKGROUND: Administration of appropriate antimicrobial therapy is one of the key factors in surviving bloodstream infections. Blood culture is currently the reference standard for diagnosis, but conventional practices have long turnaround times while diagnosis needs to be faster to improve patient care. Phenotypic methods offer an advantage over genotypic methods in that they can identify a wide range of taxa, detect the resistance currently expressed, and resist genetic variability in resistance detection. AIMS: We aimed to discuss the wide array of phenotypic methods that have recently been developed to substantially reduce the time to result from identification to antibiotic susceptibility testing. SOURCES: A literature review focusing on rapid phenotypic methods for improving the diagnosis of bloodstream infection was the source. CONTENT: Rapid phenotypic bacterial identification corresponds to Matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF), and rapid antimicrobial susceptibility testing methods comprised of numerous different approaches, are considered and critically assessed. Particular attention is also paid to emerging technologies knocking at the door of routine microbiology laboratories. Finally, workflow integration of these methods is considered. IMPLICATIONS: The broad panel of phenotypic methods currently available enables healthcare institutions to draw up their own individual approach to improve bloodstream infection diagnosis but requires a thorough evaluation of their workflow integration. Clinical microbiology will probably move towards faster methods while maintaining a complex multi-method approach as there is no all-in-one method.

Characteristics of advanced methods used for typing bacterial isolates from mastitis with particular reference to Staphylococci.

Staphylococci are very common human and animal pathogens. A variety of staphylococcal virulence determinates leads to vast range of infections. One of them is mastitis which is a common disease of the mammary glands. The incidence of this disease is widespread all over the world and depends on bacterial virulence and on prevention programs. The influence of mastitis on human health is not globally evaluated, however, in veterinary fields loses in milk production caused by bovine mastitis are a constant economic problem. One of the most important parts of the mastitis control programs is accurate diagnosis of the inflammation and characterization of the etiological factors which leads to reduction of mastitis spread. Recent reports show that staphylococci are common bacterial etiological factors of mastitis, and this paper is an overview of the diagnostic typing methods used for characterization of staphylococcal isolates. A number of different techniques available to applicate is described. Phenotypic methods to identify and to differentiate isolates or discriminate virulence factors are still in use, however, some advanced genetic methods offering higher discriminatory power are reported as more accurate. In fact, nowadays the most powerful tool on that field is next generation sequencing (NGS) of the whole genome, but its high cost and requirement of special laboratory equipment makes it hard to use for routine diagnostics. That is why standard PCR techniques-based methods, and the sequencing of particular genes, are mostly used for typing bacterial isolates. Most of these techniques are characterized by a high discriminatory power, big epidemiological concordance, and repeatable results. The presented report describes the techniques used most frequent in mastitis diagnostics related to staphylococci typing and shows their advantages and disadvantages.

Utilization of molecular and conventional methods for the identification of nontuberculous mycobacteria isolated from different water sources.

Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.

A European multi-centre External Quality Assessment (EQA) study on phenotypic and genotypic methods used for Herpes Simplex Virus (HSV) drug resistance testing.

BACKGROUND: Herpes Simplex Virus (HSV) drug resistance is a significant public health concern among immunocompromised individuals. Phenotypic assays are considered the gold standard method for detecting HSV drug resistance. However, plaque reduction assays (PRAs) are technically demanding, often with long turnaround times of up to four weeks. In contrast, genotypic tests can be performed within a few days. OBJECTIVES: The development and coordination of the first European External Quality Assessment (EQA) study to evaluate phenotypic and genotypic methods used for HSV drug resistance testing in specialised reference laboratories. STUDY DESIGN: Four HSV-1 or HSV-2 strains with different antiviral susceptibility profiles were isolated from clinical samples. Isolates were quantified by qPCR, and aliquoted in culture medium. One isolate was distributed at two dilutions to help assess assay sensitivity. The panel was distributed to five European centres with a six-week deadline for the return of phenotypic and genotypic results, together with clinical reports. RESULTS: Four out of five participating labs returned results by the deadline. Limited results were later available from the fifth lab. Phenotypic and genotypic data were largely, but not completely, concordant. An unusual resistance profile shown by one of the samples was explained by the detection of a mixed virus population after extensive further investigation by one of the centres. CONCLUSIONS: Discordant clinical outputs reflecting the diversity of phenotypic methodologies demonstrated the utility of this exercise. With emerging genotypic technologies looking to supplant phenotyping, there is a need for curated public databases, accessible interpretation tools and standardised control materials for quality management. By establishing a network of testing laboratories, we hope that this EQA scheme will facilitate ongoing progress in this area.

Challenges in the identification of Chryseobacterium indologenes and Elizabethkingia meningoseptica in cases of nosocomial infections and patients with cystic fibrosis.

Rare nonfermenting Gram-negative bacilli, such as Chryseobacterium indologenes and Elizabethkingia meningoseptica, have clinical importance in nosocomial infections and cystic fibrosis (CF), and their identification is a challenge to microbiology laboratories. Thus, the objective of this study was to verify the performance of phenotypic and mass spectrometry (matrix-assisted desorption ionization-time of flight mass spectrometry, MALDI-TOF MS) methods to identify C. indologenes and E. meningoseptica. In this context, the results obtained with phenotypic methods-namely manual biochemical and automated VITEK 2 (bioMérieux, Marcy l'Etoile, France) and Phoenix tests (Becton Dickinson (BD), San Diego, CA, USA)-and by MALDI-TOF MS-namely MALDI-TOF VITEK MS (MALDI-MS; bioMérieux) and MALDI-TOF BioTyper (MALDI-BD; BD)-of 22 isolates (blood cultures of patients with nosocomial infection (n = 15) and from patients with CF (n = 7)), initially identified as C. indologenes and E. meningoseptica, were compared. As result, using the manual phenotypic method, it was possible to identify the species level in 18/22; no identification was found in 4/22. There was a low agreement level between manual and VITEK 2 automated phenotypic methods when considering the genus level. The greatest agreement for genus-level identification occurred in MALDI-TOF MS equipment (15/22). When comparing all methods to identify the 22 isolates, there was agreement of 4/22 at the genus level and of 4/22 at the species level. In conclusion, there is low agreement level among identification methods of C. indologenes and E. meningoseptica. Although MALDI-TOF MS equipment shows a higher agreement level among them, results present low levels of confidence.

Rapid Identification of Vancomycin Resistant Enterococcus Faecalis Clinical Isolates using a Sugar Fermentation Method.

INTRODUCTION: Vancomycin Resistant Enterococci (VRE) can be found all over the world. Thus, rapid detection of the isolates could be of high importance in the treatment or prevention of the associated disease. AIM: To measure the turanose fermentation in Enterococcus faecalis clinical isolates for rapid differentiation of VRE and Vancomycin-Susceptible E. faecalis (VSE) isolates. MATERIALS AND METHODS: Forty E. faecalis samples were isolated from 200 clinical samples in Tehran Medical Center, Iran, from October 2012 to December 2012. These isolates were detected according to the standard microbial and biochemical tests. Detection of VRE isolates was originally performed by disk diffusion using 1 µg vancomycin disk, followed by Polymerase Chain Reaction (PCR) amplification of the vanA gene. Finally, the turanose consumption in 1%, 0.7% and 0.5% dilutions was detected by a phenotypic method. RESULTS: Among the 40 E. faecalis isolates, 20 vancomycin-susceptible and 20 vancomycin-resistant E. faecalis were isolated according to the disk diffusion and PCR of the vanA gene. There was a considerable difference between VRE and VSE isolates in 0.7% dilution of turanose. However, there was no significant difference between VRE and VSE in 1% and 0.5% dilutions of turanose. CONCLUSION: Since detection of VRE isolates is of high importance, especially in nosocomial infections, phenotypic methods may be highly useful for this purpose. In conclusion, our data indicate that VRE isolated from clinical samples could be distinguished from VSE isolates by turanose fermentation at dilution 0.7%.

Evaluation of Six Phenotypic Methods for the Detection of Carbapenemases in Gram-Negative Bacteria With Characterized Resistance Mechanisms.

BACKGROUND: We compared the performance of the modified Hodge test (MHT), Triton Hodge test (THT), Carba NP test (CNPt), simplified Carba NP test (CNPt-direct), blue-Carba NP test (BCT), and carbapenem inactivation method (CIM) for rapid and accurate carbapenemase detection. METHODS: The methods were evaluated by using 256 gram-negative isolates, including 197 Enterobacteriaceae (79 Enterobacter spp., 74 Klebsiella spp., 33 Escherichia coli, 10 Citrobacter spp., and 1 Serratia marcescens), 51 Acinetobacter baumannii, and 8 Pseudomonas aeruginosa strains. The collection included 117 non-carbapenemase, 18 Klebsiella pneumoniae carbapenemases (KPC) producers, 46 New Delhi metallo-ß-lactamases (NDM) producers, 11 imipenemases (IMP) producers, and 51 oxacillinases (OXA) producers, and 13 strains harboring two different carbapenemase genes. RESULTS: The specificity of the THT (91.5%) was significantly lower than other methods, each of which had 100% specificity (P<0.003). This can be attributed to the false detection of Ampler class C ß-lactamases (AmpC) carriers. The CNPt-direct and CIM yielded the highest sensitivities (P<0.003), which were comparable (92.8% vs 93.5%, P>0.999). Because of improved detection of NDM carriers, THT showed significantly higher sensitivity than the MHT (84.9% vs 75.5%, P<0.001). However, poor performances in detecting OXA still influenced the sensitivities of the CNPt (66.2%) and BCT (82.0%), as well as the MHT and THT. CONCLUSIONS: CNPt-direct and CIM demonstrated the best performance for the efficient detection of carbapenemase among the six evaluated methods. Except the MHT and THT, the detection of carbapenemase-producing Enterobacteriaceae by all the other methods was acceptable, when the OXA-type carbapenemase was not prevalent.