Clinical Laboratory Standards Institute Guidelines Research Articles

Neonatal septicemia: Blood culture bacterial isolates and their antimicrobial susceptibility pattern

Abstract Introduction: Neonatal septicemia is defined as “a clinical syndrome of bacteremia with systemic signs and symptoms of infection in the first four weeks of life”. Blood culture is the gold standard method for diagnosis of septicemia. Isolation of organism in blood culture and its antimicrobial sensitivity pattern carries a great role. Aim: To provide prevalence and antimicrobial susceptibility pattern of bacteria isolated from suspected cases of neonatal septicemia at a tertiary care hospital, Valsad. Materials and Mathods: Retrospective study is conducted at a tertiary care hospital, Valsad from January 2017 to June 2018. Processing of blood culture samples, Isolation and identification of bacteria were done using standard microbiology techniques. Antimicrobial susceptibility testing was performed using modified Kirby-Bauer disk diffusion method as per Clinical laboratory Standard Institute guideline. ATCC E. coli 25922, ATCC S. aureus 25923, ATCC P. aeruginosa 27853 were used as standard strains. Results: Out of 452 blood cultures, organisms were isolated in 127(28.09%). Commonly isolated bacteria are Coagulase Negative Staphylococci (42.52%) followed by Klebsiella spp. (18.11%), Acinetobacter spp. (15.75%), Enterococcus spp. (9.45%) and others (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa). Coagulase Negative Staphylococci strains have highest sensitivity to Vancomycin (100%), Linezolid (100%), Teicoplanin (100%), followed by Chloramphenicol (92.6%), Levofloxacin (90.7%), Cefoxitin (83.3%), and Tetracycline (77.8%). 16.7% isolates were Methicillin Resistant Staphylococci (MRS). Klebsiella spp. strain have highest sensitivity to Meropenem (73.9%), Imipenem (73.9%) and Amikacin (69.6%) followed by Chloramphenicol (65.2%), Tetracycline (60.9%) and Piperacillin Tazobactum (52.2%). 78.3% strains were Extended spectrum Beta-Lactamase (ESBL) producers. Conclusion: Antimicrobial resistances are growing among bacteria an

Multidrug-resistant and extensively drug-resistant Neisseria gonorrhoeae in Canada, 2012-2016.

BackgroundNeisseria gonorrhoeae have acquired resistance to many antimicrobials, including third generation cephalosporins and azithromycin, which are the current gonococcal combination therapy recommended by the Canadian Guidelines on Sexually Transmitted Infections.ObjectiveTo describe antimicrobial susceptibilities for N. gonorrhoeae circulating in Canada between 2012 and 2016.MethodsAntimicrobial resistance profiles were determined using agar dilution of N. gonorrhoeae isolated in Canada 2012–2016 (n=10,167) following Clinical Laboratory Standards Institute guidelines. Data were analyzed by applying multidrug-resistant gonococci (MDR-GC) and extensively drug-resistant gonococci (XDR-GC) definitions.ResultsBetween 2012 and 2016, the proportion of MDR-GC increased from 6.2% to 8.9% and a total of 19 cases of XDR-GC were identified in Canada (0.1%, 19/18,768). The proportion of isolates with decreased susceptibility to cephalosporins declined between 2012 and 2016 from 5.9% to 2.0% while azithromycin resistance increased from 0.8% to 7.2% in the same period.ConclusionWhile XDR-GC are currently rare in Canada, MDR-GC have increased over the last five years. Azithromycin resistance in N. gonorrhoeae is established and spreading in Canada, exceeding the 5% level at which the World Health Organization states an antimicrobial should be reviewed as an appropriate treatment. Continued surveillance of antimicrobial susceptibilities of N. gonorrhoeae is necessary to inform treatment guidelines and mitigate the impact of resistant gonorrhea.

Antibacterial activity of resveratrol structural analogues: A mechanistic evaluation of the structure-activity relationship

Structure-activity relationship (SAR) studies have led to significant improvement in desirable biological activity in different classes of molecules. A general consensus about the substitutions that improve the activity remains elusive in stilbene class of molecules especially in regard to antibacterial activity. Lack of this knowledge remains a major hurdle in developing stilbene based antibacterial molecules. A panel of gram positive and gram negative bacteria were employed for screening the comparative efficacy of the stilbenes. In addition, the mechanisms that contribute to the antibacterial activity were investigated and correlated to structural changes. Employing the notorious nosocomial agent S. aureus we show how changes in structure alters not only the antibacterial activity but also the underlying mechanisms. Antibacterial activity by CLSI (Clinical & Laboratory Standards Institute) guidelines, oxidative stress and membrane damage by fluorescence based methods, DNA binding by spectroscopy, DNA cleavage by gel electrophoresis, substrate efflux by efflux mutant and cell wall damage by scanning electron microscopy were investigated. Antibacterial activity varied drastically among stilbenes bearing different functional groups. The best stilbenes in terms of activity also scored higher in one or more molecular events that contribute to cell death. Stilbenes superior to resveratrol in antibacterial acitvity were identified and probable causes for better activity were also identified. Our study revealed dimerization, halogenation and hydroxy group in conjunction with methoxy group resulted in the best antibacterial molecules. Design of stilbene based drugs would be benefitted with the outcome and rationale presented in the current investigation.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley & Sons, Inc.

The application of IFCC reference methods in methodological evaluation of glycated hemoglobin measurement systems

Objective To perform a methodological evaluation study of 4 HPLC based systems and a capillary electrophoresis based system, with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) glycated hemoglobin reference method as a comparative method. Methods 40 hemolysis samples of variety concentrations were prepared. The samples were measured by IFCC reference method and 5 glycated hemoglobin testing systems, respectively and trueness verification was performed according to the Clinical & Laboratory Standards Institute (CLSI) guideline EP9-A3. Whole blood samples were used to test the systems′ precision, linearity and analytical interferences. Results The average CV of IFCC reference method results of 40 hemolysis samples was 1.4%(range from 0.2% to 2.5%). No outlier was found in the results of the 5 testing system. The slopes ranged from 0.990 2 to 1.026 7, and intercepts from -0.152 6 mmol/mol to + 0.151 2 mmol/mol, squared correlation coefficient from 0.996 2-0.997 1, biases at two medical decision level were less than 0.3% HbA1c. Within-laboratory precisions were less than 2% (NGSP unit). Bias between all test results and predicted results were less than 5%. High concentration of glucose showed certain interference to glycated Hemoglobin tests but had little influence in clinical practice. Conclusions The results of the 5 testing system are comparable to the IFCC reference method, the results of precision and linearity evaluation meet the requirements of related guidelines.(Chin J Lab Med, 2018, 41: 821-826) Key words: Hemoglobin A, glycosylated; Reference values; Reference standards; Chromatography, high pressure liquid; Chromatography, ion exchange; Electrophoresis, capillary; Evaluation studies

Evaluation of In-vitro Antimicrobial Potential of Daphne retusa Hemsl. Against Human Pathogenic Bacteria and Fungi.

Antimicrobial drug resistance is an emerging problem, which leads to a failure in the control of infectious diseases thereby, adversely affecting patient care and reducing effective management of infectious diseases globally. Thus, search for new and more effective alternatives is needed. Daphne retusa Hemsl. (Daphne) has medicinal values and is reported to be widely used in curing a variety of human ailments. Current study assesses in-vitro antimicrobial activity of the crude extract of D.retusa (whole plant) and its derived fractions against clinically isolated human pathogenic bacteria and fungi. Whole plant of D.retusa was powder dried and then extracted with methanol (E1). The resultant was fractionated to give Chloroform fraction (E2), Butanol fraction (E3) and Ethyl acetate fraction (E4). The crude extract and derived fractions were assessed for antimicrobial and antifungal activity by using agar well diffusion method and their MICs were found following Clinical and Laboratory Standard Institute (CLSI) guidelines. Our study shows that D.retusa has very good inhibitory action against different bacterial and fungal strains. All of the extracts were active against almost every microorganism used in the study. E2 has the maximum percent of inhibition against bacterial growth while E1 has themaximum percent of inhibition against fungal growth. Streptococcus pneumonia was the most susceptible bacteria while among fungi, Gongronella butleri showed highest susceptibility. Results justify the use of D. retusa in the treatment of microbial infections. For the development of a novel antibiotic, the crude extract and its derived fractions need further exploration; with emphasis to isolate and identify the active constituents that are responsible for antibacterial and antifungal activity.

ANTIBACTERIAL POTENTIAL OF L-TRYPTOPHAN ON EXTENDED-SPECTRUM BETALACTAMASE PRODUCING ESCHERICHIA COLI STRAINS: A PATHOLOGICAL PROMISING APPROACH

Objective: The main aim of this study was to determine the extended-spectrum beta-lactamase (ESBL) production among 83 isolates of Escherichia coli as well as the antibacterial effect of a novel compound, L-Tryptophan Schiff base, on 10 different ESBL positive strains of E. coli. Methods: Phenotypic ESBL activity in E. coli was confirmed by combined disc diffusion test according to clinical laboratory standard institute guidelines. Phenotypically positive ESBL clinical isolates were selected for molecular screening for synchronized detection of blaCTX-M-15 gene. Antibacterial activity of L-Tryptophan Schiff base was evaluated against ESBL positive isolates. The effect of L-Tryptophan Schiff base on a polymerase chain reaction (PCR) amplified a product of CTX-M-15 gene was also evaluated.Results: Antibiotic susceptibility screening showed resistance of ESBL positive isolates in the range of 18–96%. Disc diffusion test for phenotypic ESBL detection revealed that 99% (83/83) of isolates showed resistance to third and fourth generation cephalosporins (ceftazidime, cefotaxime, and cefepime) including ampicillin. L-Tryptophan Schiff base showed antimicrobial effect on E. coli. Molecular analysis for ESBL detection showed that 60% of strains were positive for blaCTX-M-15 gene. L-Tryptophan Schiff base also had downregulating effect on CTX-M-15 gene.Conclusion: This study presented an approach toward finding a suitable drug to reduce the serious infections caused by ESBL positive microorganisms. Phenotypic as well as molecular characterization was performed to get knowledge about the overall behavior of ESBL positive isolates of E. coli. L-Tryptophan Schiff base showed good antibacterial properties against ESBL positive isolates and a downregulating effect on PCR amplified product of CTX-M-15.

Order of draw practices in venous blood sampling at clinical biochemistry departments in the Danish health care system

BackgroundDeviation in blood collection procedures is a central source of preanalytical variation affecting overall analytical and diagnostic precision. The order of draw of venous sampling is suspected to affect analytical results, in particular for coagulation analysis. Here we compare the procedures in venous blood sampling among clinical biochemistry departments to assess the uniformity of order of blood draw and adherence to international guidelines in the Danish health care system. MethodsWe collected venous order of draw procedures from 49 clinical biochemistry departments at 22 public hospitals in Denmark. Procedures were compared to the international guidelines fromthe Clinical Laboratory Standards Institute (CLSI) and World Health Organization (WHO), and assessed in relation to department ISO 15189:2012 accreditation. ResultsWe observed seven different order of draw procedures related to citrate, serum, heparin, and EDTA tubes, and the use of discard tubes in relation to coagulation assays. 31 departments (63.3%) were found to adhere to CLSI and WHO guidelines. A majority of departments instructs the use of discard tubes before collection for coagulation assays in citrate tubes (44 departments; 89.8%). The citrate tube was the first sample tube to be drawn for most departments (35 departments; 75.5%); and the preferred order of non-citrate tubes was serum-heparin-EDTA (36 departments; 73.5%). Adherence to the CLSI and WHO guidelines was not associated with department ISO 15189:2012 accreditation (p = .57). ConclusionsVenous order of draw procedures is diverse at Danish clinical biochemistry departments and show moderate adherence to international guidelines.

In vitro antimicrobial activity of monensin against common clinical isolates associated with canine otitis externa.

Antimicrobial resistance and antimicrobial stewardship are of ever-increasing importance in veterinary medicine. Multidrug-resistant infections of the canine skin and ear continue to emerge, but the use of antibiotic classes of critical importance to human medicine may not represent good antimicrobial stewardship. Repurposing of old drugs that are not used in human medicine is one approach that addresses both these issues. In this study, the minimal inhibitory concentration (MIC) of monensin for 111 bacterial and yeast canine otitis isolates was determined using microdilution methodology according to Clinical Laboratory Standards Institute (CLSI) guidelines. Monensin was effective against all Gram-positive bacteria including the multidrug-resistant staphylococcal strains with MICs ranging from 1 to 4 μg/ml, but lacked antimicrobial activity against Gram-negative bacteria and yeast isolates. Monensin has potential to be incorporated as one of the main components in an otic formulation.

Antimicrobial Resistant Enterococcus faecium, Enterococcus faecalis, and Other Enterococcus Species Isolated From Foal Feces in Japan

Antimicrobial resistant (AMR) Enterococci are an emerging problem for human and animal health. Few studies have focused on the detection of AMR in foals; therefore, we conducted this study to investigate the occurrence of AMR Enterococci in foal feces in Japan. A total of 72 fecal samples from healthy foals were collected from seven horse stables, in Hokkaido Prefecture, from June 2015 to January 2016. Enterococci were isolated and identified using EF Agar Base selective media, and the species confirmed using MALDI Biotyper systems. The minimum inhibitory concentration of nine antimicrobial agents for the isolates was determined based on the Clinical Laboratory Standard Institute guidelines. A total of 183 isolates of Enterococci were identified as Enterococcus faecium 54.1% (99/183), Enterococcus faecalis 16.4% (30/183), and other species 29.5% (54/183). The highest occurrence of E. faecium antibiotic resistant was to erythromycin at 55.6% (55/99), followed by enrofloxacin, kanamycin, and oxytetracycline, at 30.3%, 7.1%, and 4.0%, respectively. In contrast, E. faecalis isolates showed higher resistant to oxytetracycline 76.7%, kanamycin 46.7%, gentamycin 30.0%, chloramphenicol 16.7%, lincomycin 30.0%, and tylosin 30.0%. E. faecium highly resistant to erythromycin and enrofloxacin but lowly resistant to gentamycin and tylosin, and E. faecalis was highly resistant to kanamycin, oxytetracycline, and lincomycin. Antimicrobial resistant E. faecalis (30.0%) and E. faecium (4.0%) isolates show multidrug resistant to at least three different classes of antibiotics. Our study indicated that foal feces may act as a source of AMR genes for Enterococci that may be transmitted to other animals, humans, and the environment.

UPLC–MS/MS assay validation for tacrolimus quantitative determination in peripheral blood T CD4+ and B CD19+ lymphocytes

Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC–MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid–liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters® Acquity™ UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters® MassTrak TDM C18 (2.1 × 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0–5.2 ng/mL in PBMC, 0–5.0 ng/mL in T CD4+ Lymphocytes and 0–5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at −30 °C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/106 cells in PBMC, from 0.081 to 1.432 ng/106 cells in T CD4+, and from 0.197 to 1.564 ng/106 cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.

CLSI-based transference and verification of CALIPER pediatric reference intervals for 29 Ortho VITROS 5600 chemistry assays

BackgroundEvidence-based reference intervals (RIs) are essential to accurately interpret pediatric laboratory test results. To fill gaps in pediatric RIs, the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) project developed an age- and sex-specific pediatric RI database based on healthy pediatric subjects. Originally established for Abbott ARCHITECT assays, CALIPER RIs were transferred to assays on Beckman, Roche, Siemens, and Ortho analytical platforms. This study provides transferred reference intervals for 29 biochemical assays for the Ortho VITROS 5600 Chemistry System (Ortho). MethodsBased on Clinical Laboratory Standards Institute (CLSI) guidelines, a method comparison analysis was performed by measuring approximately 200 patient serum samples using Abbott and Ortho assays. The equation of the line of best fit was calculated and the appropriateness of the linear model was assessed. This equation was used to transfer RIs from Abbott to Ortho assays. Transferred RIs were verified using 84 healthy pediatric serum samples from the CALIPER cohort. ResultsRIs for most chemistry analytes successfully transferred from Abbott to Ortho assays. Calcium and CO2 did not meet statistical criteria for transference (r2<0.70). Of the 32 transferred reference intervals, 29 successfully verified with approximately 90% of results from reference samples falling within transferred confidence limits. Transferred RIs for total bilirubin, magnesium, and LDH did not meet verification criteria and are not reported. ConclusionsThis study broadens the utility of the CALIPER pediatric RI database to laboratories using Ortho VITROS 5600 biochemical assays. Clinical laboratories should verify CALIPER reference intervals for their specific analytical platform and local population as recommended by CLSI.

Antimicrobial Susceptibility Profiles of Gram-Negative Bacteria Causing Infections Collected Across India during 2014–2016: Study for Monitoring Antimicrobial Resistance Trend Report

Background: The emergence of antibiotic resistance among bacterial pathogens in the hospital and community has increased the concern to the health-care providers due to the limited treatment options. Surveillance of antimicrobial resistance (AMR) in frequently isolated bacterial pathogens causing severe infections is of great importance. The data generated will be useful for the clinicians to decide empiric therapy on the local epidemiological resistance profile of the antimicrobial agents. This study aims to monitor the distribution of bacterial pathogen and their susceptibility pattern to the commonly used antimicrobial agents. Materials and Methods: This study includes Gram-negative bacilli collected from intra-abdominal, urinary tract and respiratory tract infections during 2014–2016. Isolates were collected from seven hospitals across India. All the study isolates were characterised up to species level, and minimum inhibitory concentration was determined for a wide range of antimicrobials included in the study panel. The test results were interpreted as per standard Clinical Laboratory Standards Institute guidelines. Results: A total of 2731 isolates of gram-negative bacteria were tested during study period. The most frequently isolated pathogens were 44% of Escherichia coli (n = 1205) followed by 25% of Klebsiella pneumoniae (n = 676) and 11% of Pseudomonas aeruginosa (n = 308). Among the antimicrobials tested, carbapenems were the most active, followed by amikacin and piperacillin/tazobactam. The rate of extended-spectrum beta-lactamase (ESBL)-positive isolates were ranged from 66%–77% in E. coli to 61%–72% in K. pneumoniae, respectively. Overall, colistin retains its activity in > 90% of the isolates tested and appear promising. Conclusion: Increasing rates of ESBL producers have been noted, which is alarming. Further, carbapenem resistance was also gradually increasing, which needs much attention. Overall, this study data show that carbapenems, amikacin and colistin continue to be the best agents available to treat drug-resistant infections. Thus continuous monitoring of susceptibility profile of the clinically important Gram-negative pathogens is of great importance to guide effective antimicrobial therapy.

Prevalence of nasal colonization of methicillin-resistant Staphylococcus aureus among schoolchildren of Barabanki district, Uttar Pradesh, India.

Introduction:The study aimed to determine the prevalence of nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA), the minimum inhibitory concentration (MIC) of oxacillin and vancomycin, inducible clindamycin resistance, and antimicrobial resistance pattern of S. aureus among children of Barabanki district, Uttar Pradesh, India.Materials and Methods:School-going children of age group of 5–15 years were identified and selected according to the inclusion and exclusion criteria. Two nasal swabs were collected from each child as per the Centers for Disease Control and Prevention guidelines and transported to laboratory. Swabs were cultured on mannitol salt agar and 5% blood agar and incubated for 18–24 h at 37°C. Identification was done as per routine laboratory protocol. Detection of MRSA was done through cefoxitin 30 μg discs and D-zone test. Antibiotic susceptibility pattern of S. aureus by Kirby–Bauer disc diffusion method along with MIC for oxacillin and vancomycin was performed simultaneously according to Clinical Laboratory Standards Institute guidelines.Results:Out of 300 children, 140 (46.67%) were found to be nasal carriage for S. aureus, among which MRSA was found to be 23 (7.67%). All S. aureus and MRSA isolates were sensitive to vancomycin with MIC <2 μg/ml, whereas 23 S. aureus were found resistant to oxacillin with MIC value >4 μg/ml. Resistance to penicillin and co-trimoxazole was highest, whereas all were sensitive to linezolid. MRSA showed 100% susceptibility to linezolid, followed by gentamicin (91.4%) and tetracycline (87%).Conclusion:With the risk involved in transmission of infection, steps for identifying the carriers and its eradication should be carried out. Rational use of antibiotics should be given preference too.

Epidemiology, clinical profile and role of rapid tests in the diagnosis of acute bacterial meningitis in children (aged 1-59 months).

To study the epidemiology, clinical profile, and the role of rapid tests in the diagnosis of acute bacterial meningitis (ABM) in children (1-59 months). A total of 250 cerebrospinal fluid (CSF) and 187 blood samples received from clinically suspected cases of ABM were processed based on standard microbiological protocols. CSF samples were also subjected to antigen and nucleic acid detection. Antibiotic susceptibility testing was done according to the Clinical Laboratory Standards Institute guidelines. Children were also evaluated for outcomes and were followed up until 6 months after discharge. Eighty one cases were reported to be having clinically confirmed ABM, out of which group B Streptococcus was the most common pathogen detected in 49.3% (40) patients followed by Streptococcus pneumoniae, Staphylococcus aureus, Hemophilus influenzae type b, Escherichia coli, Klebsiella pneumoniae, and Neisseria meningitidis ACYW135 in 23.4% (19), 7.4% (6), 6.1% (5), 6.1% (5), 6.1% (5), and in 1.2% (1) patients, respectively. Complications were observed in 54.3% of the children. A follow-up of 6 months after discharge was possible in 39.5% (32) patients among whom sequelae were recorded in 93.7% (30) patients. ABM remains a major cause of neurological sequelae worldwide. Although culture is the gold standard test for its detection, the investigation takes a longer time and the results are influenced by prior antimicrobial therapy. In such cases, rapid tests aid in the early diagnosis of ABM for instituting appropriate management.

AACC Academy's Pain Management LMPG: Verification of Drug Dosing with Quantitative Urine Drug Testing?

Clinical practice guidelines have become a mainstay for streamlining medical practices and function to ensure uniformity. Clinical practice guidelines are relevant in the world of laboratory medicine, given the large number of medical decisions that are based on the result of a clinical laboratory test. The first set of guidelines was created in 1995 by the National Academy of Clinical Biochemistry (now the AACC Academy) and was called “Standards of Laboratory Practice.” The topic was the laboratory support of thyroid disease, and the guidelines were first published as a series of papers (1) and then widely distributed as a monograph. Later, guidelines were produced under the label “Laboratory Medicine Practice Guidelines” (LMPGs). In this issue of JALM , the Academy's latest LMPG addresses laboratory tests to monitor drug therapy in pain management patients (2). As an author of several other LMPGs, including one on laboratory tests to support cases of drug poisoning (3), a coauthor of a recently released toxicology guideline, the Clinical Laboratory Standards Institute Guideline on Toxicology and …

Biofilm production by Staphylococcal isolates from chronic osteomyelitis and orthopedic device-related infections in a tertiary care centre, Imphal

Background: Microorganisms growing in a biofilm are associated with chronic, recurrent infections, and health problems for patients with indwelling devices which are difficult to treat with antimicrobial agents. Staphylococcus species are the most common microorganisms isolated from these patients. This study was conducted to detect biofilm production in Staphylococcal isolates from orthopedic device-related infections (ODRI) and chronic osteomyelitis (CO) and correlation of antimicrobial susceptibility pattern between biofilm producers and nonbiofilm producers. Materials and Methods: A cross-sectional study was carried out at the Department of Microbiology in a tertiary care center after the Institutional Ethics Committee approval, from October 2014 to September 2016. Detection of biofilm was performed by Congo Red Agar (CRA), tube method (TM), and tissue culture plate (TCP) methods. Antimicrobial susceptibility test was performed by Kirby–Bauer disk diffusion method, and minimum inhibitory concentration for oxacillin and vancomycin was determined according to the Clinical Laboratory Standard Institute guidelines. Results: A total of 51 clinical isolates, consisting of 45 Staphylococcus aureus and 6 coagulase-negative Staphylococci (CoNS), were isolated from 47 CO and 4 ODRI patients of which 32 were methicillin-resistant S. aureus and 2 MRCONS. Biofilm production was detected in 22 isolates by TCP, 18 by TM, and 13 by CRA methods. Fifty percent ODRI isolates and 42.5% CO isolates were biofilm producers. The biofilm producers have lower antimicrobial sensitivity pattern in all except vancomycin. Conclusion: Biofilm producers were detected from 43.1% of total isolates with higher antimicrobial resistance pattern.

Prevalence and Antimicrobial Susceptibility Patterns of ESBL, AmpC and Carbapenemase-producing Enterobactericeae Isolated from Hospitalized Patients in Azerbaijan, Iran.

The current study was conducted to determine prevalence and antimicrobial susceptibility patterns, ESBL, AmpC and carbapenemase- producers among clinical isolates of Enterobacteriaceae. Three hundred and seven non-duplicative clinical isolates were collected from hospitalized patients in five medical centers in Azerbaijan, Iran. Initial screening for β-lactamase production was performed using disc agar diffusion according to Clinical Laboratory Standards Institute (CLSI) guidelines. Phenotypic confirmatory tests was done using total ESBL/AmpC confirm kit, KPC/MBL and OXA-48 confirm kit according to manufacturer's instructions. The overall prevalence of ESBL, AmpC, and carbapenemase-producing strains were 42.7% (131/307), 14.0%(43/307) and 4.9% (15/307), respectively. The prevalence of ESBLs was 38.35%, 64.9% and 35.7% for E. coli, K. pneumoniae and E. cloacae, respectively. Carbapenemase activity was only observed among 15 K. pneumoniae isolates and detected phenotypes include MBL (9/57, 15.8%), KPC (4/57, 7.0%), and OXA-48 (2/57, 3.5%). Fourteen out of 307 isolates (4.6%) were recognized to have ESBL or AmpC hyper-producer with decreased cell wall permeability phenotype. All 4 shigella strains were positive for ESBL and 4 isolates of P. mirabilis, 2 isolates of M. morganii and 1 seratia spp. were detected as AmpC producer. The only C. freundii strain isolated was positive for both ESBL and AmpC. This study reveals high prevalence of multidrug-resistant (MDR) β-lactamase-producing Enterobactericeae reaching 89.5%. Imipenem and meropenem showed potent antibacterial activities against all MDR β-lactamase-producers except for carbapenemase producers. After carbapenems, amikacin, piperacillin/tazobactam and amoxicillin/clavulanic acid were second the most effective drugs against β-lactamase-producing E. coli isolates.

Outbreak of Pichia kudriavzevii fungaemia in a neonatal intensive care unit.

Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, Pichia kudriavzevii (a teleomorph of Candida krusei). To the best of our knowledge, this is the first report of neonatal sepsis due to P. kudriavzevii. Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed. Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as P. kudriavzevii and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices. This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.

Fecal Carriage of Carbapenem-Resistant Enterobacteriaceae and Risk Factor Analysis in Hospitalised Patients: A Single Centre Study from India

Purpose: Carbapenem-resistant Enterobacteriaceae (CRE) have emerged and disseminated widely causing a variety of infections. In India, the carriage of CRE in hospitalised patients has not been well-studied. Therefore, we conducted the present study to observe gut carriage rate of CRE in patients admitted to our tertiary care hospital. Methods: A total of 232 faecal swabs collected from consecutive stool samples from admitted patients were inoculated on ChromID extended spectrum β-lactamase plates and members of Enterobacteriaceae family were subjected to antibiotic susceptibility as per the Clinical Laboratory Standards Institute guidelines. Polymerase chain reaction for blaVIM, blaKPC, blaIMP and blaNDM-1 genes was performed. CRE was identified if the isolates showed resistance to either imipenem or meropenem or showed the presence of resistant genes. Risk factors of patients with or without CRE colonisation were also analysed. Results: A total of 232 faecal swabs yielded 252 Enterobacteriaceae isolates, of which 49 isolates from 42 patients showed the presence of CRE (occurrence 42/232; 18.1%); 27 isolates from 22 patients carried blaNDM-1, whereas 20 isolates from 17 patients possessed blaVIM gene. No isolate was positive for blaKPC and blaIMP genes. The CRE was common in both intensive care units (38.4%) and wards (46%) which may reflect the excessive use of broad-spectrum antibiotics in both these settings. The CRE was also found to have a significantly higher antimicrobial resistance as compared to non-CRE isolates. The logistic regression analysis of significance showed the presence of any indwelling device (P = 0.049) and nasogastric tube (P = 0.043) as independent risk factors for acquiring gut colonisation. Conclusions: The study is the first from India to show high CRE carriage in patients admitted to a tertiary care centre and emphasises the need of strict antimicrobial stewardship implementation in hospitals to prevent dissemination of multidrug-resistant CRE.