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2019-Standard Operating Procedures Standard Operating Procedures Bacteriology Antimicrobial Resistance Surveillance and Research Network 2nd Edition, 2019 Indian Council of Medical Research New Delhi, India Prof. Balram Bhargava Secretary, Department of Health Research (DHR) and Director General, Indian Council of Medical Research Published in 2015, 1st Edition 2019, 2nd Edition Printed in 2019 Dr. Neeraj Tandon: Head (Publication & Information) Published by the Division of Publication and Information on behalf of the Secretary, DHR and Director General, ICMR, New Delhi Printed by M/s. Royal Offset Printers, A-89/1, Naraina Industrial Area, Phase-I, New Delhi-110028 Foreword This Standard Operating Procedure (SOP) manual is intended to assist as a reference material for routine laboratory activities of ICMR's 'Antimicrobial Resistance Surveillance and Research Network' (AMRSN). This edition of SOP embodies the continued efforts of ICMR to provide clinically relevant and practical document for the standardized in vitro susceptibility testing (AST) of bacterial pathogens. The manual guide the expected and required procedures needs to be followed by laboratory personnel while handling bacterial pathogens and performing AST to enhance the operational performance. I am glad that ICMR’s national network on anti-microbial drug resistance is continuously thriving for containment of AMR and providing in-depth understanding of clonality of drug resistant pathogens and their transmission dynamics. The manual describes the well accepted methods of specimen collection, transport, culture, identification and anti-microbial susceptibility testing on clinically important gram positive and gram negative bacteria. The major revisions include procedures for tissue samples collection, identification of Streptococcus spp. (beta and alpha hemolytic) and Streptococcus pneumoniae, automated methods of Minimum Inhibitory Concentration (MIC) testing and updated zone diameters and MIC breakpoints. This edition has been updated for phenotypic and genotypic methods (molecular mechanism of resistance) of AST and External Quality Assurance Scheme (EQAS) in a concise and easy manner. The manual has been improved and updated to be in sync with CLSI standard documents. Reference to any commercial method or equipment does not mean endorsement of ICMR, this is only for the purpose of this research study. I applaud the diligent efforts of editorial board members and ICMR team. I am hopeful that users will be benefited immensely from this manual and it will meet its intended objectives. I am optimistic that it will further evolve for the clinical as well as research purposes through periodic revisions and updates. I convey my best wishes to all. Prof. Balram Bhargava Secretary, DHR & Director General, ICMR CONTENTS Sr. No. Title Page No. 1. Abbreviations vii-viii 2. Distribution list (Controlled copies) ix 3. Amendment sheet x 4. Chapter 1: General guidelines 1-13 5. Chapter 2: Specimen collection, transport & processing 15-47 Blood CSF Body fluids Ocular specimens Respiratory specimens Pus Urine Fecal specimen Tissue 6. Chapter 3: Identification of isolates 49-77 Enterobacteriaceae Salmonella Stenotrophomonas maltophilia, Burkholderia cepacia complex Pseudomonas Acinetobacter Staphylococci Enterococci Fecal isolates Streptococcus sp (beta and alpha hemolytic) Streptococcus pneumoniae 7. Chapter 4: Antimicrobial Susceptibility Testing 79-103 Definitions Disc diffusion testing ATCC control strains Preparing antibiotic discs in-house Minimum Inhibitory Concentration (MIC) testing Zone diameters and MIC breakpoints 8. Chapter 5: Special Tests (Phenotypic) 105-113 Carba-NP test (For Enterobacteriaceae and Pseudomonas spp.) Modified carbapenem inactivation method (mCIM and eCIM); (For Enterobacteriaceae and Pseudomonas spp.) D-test for inducible clindamycin resistance Vancomycin screen agar for S. aureus and Enterococcus spp. MIC for vancomycin by broth micro dilution method Detection of heteroresistant vancomycin intermediate Staphylococcus aureus (hVISA) population analysis profile/area under curve (PAP/AUC) analysis Combination antimicrobial testing to evaluate the best combination of drugs for MRSA Detection of over-expression of efflux pumps MIC 9. Chapter 6: Quality control (QC) 115-118 Reference strains for QC Storing and testing QC strains Frequency of testing Quality control of media 10. Chapter 7: Molecular mechanisms of antimicrobial resistance 119-143 Enterobacteriaceae Typhoidal Salmonella: Salmonella Typhi Resistance gene detection in Pseudomonas spp. & Acinetobacter spp., using multiplex-PCR Resistance gene detection in Shigella spp. by PCR Staphylococcus species Enterococcus spp. 11. Chapter 8: External Quality Assurance Scheme (EQAS) 145-163 12. Surveillance of carriage of MDR Enterobacteriaceae in general 165-166 population 13. Surveillance of antimicrobial resistance in environmental isolates 167-168 14. Annexures I: Map of Chandigarh with proposed sites for surveillance 171 II: Biochemical reactions 172-183 III. Preparation of bacteriological media and reagents 184-193 IV. Flowchart of Workflow for Enterobacteriaceae 194 15. References 195-199 ABBREViations AST Antimicrobial susceptibility testing ATCC American Type Culture Collection BAL Bronchoalveolar lavage BAP Blood Agar Plate BCC Burkholderia cepacia complex BHIB Brain heart infusion broth BPMM Biphasic McConkey medium BSL Biosafety level CAPD Continuous ambulatory peritoneal dialysis CHOC Chocolate agar CLSI Clinical and Laboratory Standards Institute CoNS Coagulase negative staphylococci CLABSI Central Line Associated Blood Stream Infection CRBSI Catheter related blood stream infection CTA Cystine trypticase agar DD Double disk DS Double strength EDS EDTA disk synergy ESBL Extended spectrum β-lactamases ETA Endotracheal aspirate HLAR High level aminoglycoside resistance HTM Haemophilus test medium IAMM Indian Association of Medical Microbiologists MAC McConkey agar MBC Minimum bactericidal concentration MBL Metallo beta-lactamases MHA Müeller Hinton agar MHB Mueller Hinton broth MH-SB Mueller Hinton sheep blood agar MHT Modified Hodge test MIC Minimum inhibitory concentration MRSA Methicillin resistant Staphylococcus aureus NA Nutrient agar NB Nutrient broth NS Non-susceptible OF Oxidation-fermentation test OLB Open lung biopsy ONPG Ortho-nitrophenyl-β-D galactopyranoside PCN Percutaneous nephrostomy PCR Polymerase chain reaction PDA Phenylalanine deaminase PMNs Polymorphonuclear cells PPA Phenylpyruvic acid PPE Personal protective equipment QC Quality control RCM Robertson’s cooked meat SBP Spontaneous bacterial peritonitis SEC Squamous epithelial cells SPS Sodium polyanetholsulfonate TCBS Thiosulfate citrate bile salt sucrose agar TSA Trypticase soya agar TSB Trypticase soya broth TSBA Trypticase soya blood agar UTI Urinary tract infection YLO Yeast-Like Organisms DISTRIBUTION LIST (CONTROLLED COPIES) S. No. Institutes 1 Indian Council of Medical Research (ICMR), New Delhi ICMR Nodal Centers 2 All India Institute of Medical Sciences (AIIMS), New Delhi 3 Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), 4 Puducherry 5 Christian Medical College (CMC), Vellore ICMR Regional Centres 6 Mahatma Gandhi Institute of Medical Sciences (MGIMS), Wardha, Sevagram 7 Tata Medical Center (TMC), Kolkata 8 Sir Ganga Ram Hospital, (SGRH), New Delhi 9 Apollo Hospitals, Chennai 10 P.D. Hinduja National Hospital, Mumbai 11 Armed Forces Medical College (AFMC), Pune 12 King George’s Medical University (KGMU), Lucknow 13 All India Institute of Medical Sciences (AIIMS), Bhopal Lokmanya Tilak Municipal Medical College & General Hospital (LTMMC & GH), 14 Mumbai 15 Assam Medical College & Hospital (AMCH), Assam 16 Nizam’s Institute of Medical Sciences (NIMS), Hyderabad 17 Kasturba Medical College (KMC), Manipal, Karnataka 18 Institute of Postgraduate Medical Education & Research (IPGME & R), Kolkata 19 Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Soura, Srinagar 20 All India Institute of Medical Sciences (AIIMS), Jodhpur 21 Regional Institute of Medical Science, Manipur AMENDMENT SHEET S. Page Clause Date of Amendment Reason Signature Signature Lab No. No No amendment made SR Director General Guidelines CHAPTER 1 General Guidelines 1. Introduction This is a comprehensive standard operative procedure manual for all types of specimens received in a clinical bacteriology diagnostic laboratory serving a tertiary care hospital. The manual has been compiled by referring to international protocols customized to the needs and the infrastructure already available in India or infrastructure that can be achieved by upgradation. Both conventional and automated procedural alternatives are included. It is intended that all participating laboratories, including Nodal Centers and the Regional Center laboratories, will strictly adhere to the procedures. The manual has been organized to place each part of the procedure together, including sample collection, specimen processing, supplies, quality control (QC), and step-by-step testing procedure. This will allow the user to see an overview of the entire procedure together. Guidelines for specimen collection and transport can be separately made available to the collection points and those for processing are to be made available in the processing laboratories. All the laboratories must isolate, identify to species level and carry out susceptibility tests of significant bacterial isolates as per guidelines
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