Quality Control of Antimicrobial Susceptibility Tests

Transcription

1 Original Article GCSMC J Med Sci Vl (V) N (II) July-December 2016 Quality Cntrl f Antimicrbial Susceptibility Tests Falguni V. Patel, * Mahendra Vegad,** Shah P.K.,*** Kruti Tanna**** Abstract : Backgrund: Susceptibility test may be perfrmed in the clinical labratry fr tw main purpses. 1)T guide the clinician in selecting the best antimicrbial agent fr an individual patient. 2) T accumulate epidemilgical infrmatin n the resistance f micrrganisms f public health imprtance within the cmmunity. Aims & Objectives: T define variables affecting antimicrbial susceptibility testing in disc diffusin methd in micrbilgy labratry, B.J. Medical Cllege, Ahmedabad, Gujarat. Materials & Methds: This is an bservatinal study which was dne by using disc diffusin methd. Ttal 600 Antimicrbial Susceptibility tests f st th weekly Quality Cntrl(QC) were bserved frm 1 September 2014 t 30 April Out f ttal 600 Antimicrbial Susceptibility tests f weekly QC,200 tests were f E. cli 25922, 200 tests were f S. aureus and 200 were f P. aeruginsa Results: Out f 600 Antimicrbial Susceptibility Tests f weekly QC, abnrmal results were fund with 22 (3.7 % ) tests and unaffected tests were 578 (96.4 %). Variables affecting Antimicrbial Susceptibility Testing (AST) were in 22 tests. Out f them, factrs cntributed by media were 5 (0.8 %), antimicrbial disc were 3(0.5 %), inculums were 5(0.8%), envirnmental factrs were 3 (0.5%), incubatin were 2(0.33%), bservatinal factrs were 2 (0.33%) & equipment prblems in 2(0.33%). Cnclusin: Internal quality assessment is a useful cmplementary apprach t external quality assessment and may detect prblem areas nt highlighted by ther cntrl methds. Educatin, accuracy and training are an imprtant part f the quality assurance prcess. Key wrds: Antimicrbial Susceptibility Test, Quality Cntrl Intrductin : Mst f the clinically imprtant bacteria causing infectins in humans are capable f exhibiting resistance t antimicrbial agents cmmnly used fr the treatment. Thus, the reprt prduced by clinical micrbilgy labratry fr the physician, als includes rganism's susceptibility prfile t different (1) antimicrbials alng with its identificatin. Antimicrbial susceptibility testing (AST) is perfrmed n bacteria which are islated frm clinical specimens t determine if they are killed, inhibited r unaffected by the antimicrbial drugs that may be ptential chices fr therapy. Althugh the imprtance f antimicrbial susceptibility testing is well established, the prcedure itself is very sensitive t changes in the envirnment and test cnditins. Therefre, it is crucial that each variable in the prcedure shuld be standardized and carefully cntrlled. Owing t the numerus variables that may (2) affect the results, rigrus quality cntrl is f utmst imprtance fr susceptibility testing. Prperly perfrmed quality cntrl wuld aid in prviding accurate, reprducible and timely results. Materials and Methds: This is an bservatinal study which was dne by using disc diffusin methd. Ttal 600 Antimicrbial Susceptibility tests f weekly st th QC were bserved frm 1 September 2014 t 30 April Out f the ttal 600 Antimicrbial susceptibility tests, 200 tests were f E. cli 25922, 200 tests were f S. aureus and 200 were f P. aeruginsa Figure 1: Antimicrbial Sensitivity Testing f the QC strains. * Resident, ** Prfessr & Head, *** Prfessr, **** Assistant Prfessr, Micrbilgy Department, B.J. Medical Cllege, Ahmedabad. Crrespndence t : drfalgunimicr@gmail.cm P.aeruginsa S.aureus E.Cli :: 89 ::

2 Patel F V et al: Antimicrbial Susceptibility Tests We have used Agar disc diffusin methd taking int accunt the fllwing particulars: Table 1 : Specificatins fr perfrming AST Medium Mueller Hintn 4 mm thickness Agar ph : 7.2 t 7.4 Antibitic Strage discs temperature -20 C minimum 8 Inculum McFarland bacteria/ml Incubatr Temperature 37 C Prcedure: Atmsphere Ambient air Figure 2(a) &2(b): Mueller Hintn Agar medium & McFarland Standard (a) Mueller Hintn agar (b) McFarland Standard 0.5 We used well-islated, hur ld clnies f the rganism which was transferred t a sterile saline tube with a turbidity f 0.5 McFarland. Then Mueller Hintn agar was inculated by swabbing in three different directins frming a Lawn f grwth and filter paper discs impregnated with antimicrbial agents were placed n the agar. And finally it was inverted and incubated fr hurs at 37 C. During incubatin, drug diffuses int agar. Depending n the susceptibility f rganisms, areas f n grwth frm a zne f inhibitin. Znes are measured t determine whether the rganism is susceptible, intermediate, r resistant t the drug. Figure 3: AST f test rganism shwing znes f inhibitin. Amngst the tests indicating unacceptable perfrmances, the surces f the errrs were investigated with help f daily recrds f: Refrigeratr temperature, deep freeze temperature and incubatr temperature. Thickness and ph f media. Antimicrbial disc expiry date recrds. Zne diameter f weekly QC strain. Over grwths, scanty grwths and mix grwths. Sterility testing f petridish,mh agar,swab, bi safety cabinet & incubatr. Humidity & envirnment temperature. QC f MH agar plate. Errrs in media, antimicrbial discs, inculum, incubatin,equipments, bservatin and envirnmental factrs were nted. Result: In the study,we have dne a ttal f 600 Antimicrbial Susceptibility Tests f weekly QC f E. cli 25922, S. aureus and P.aeruginsa Out f 600 tests, 578 (96.4%) tests were unaffected while 22(3.6%) tests were affected due t varius variable factrs. In them, media was the cntributing factr in 5 (0.8%) tests, in 3 (0.5%) tests prblems were fund in antimicrbial discs, inculums were cntributing factrs in 5 (0.8%) tests, 3 (0.5 %)tests were affected by envirnmental factrs, bservatinal errr was affecting 2 (0.33%) tests and prblems in equipment and incubatin were fund in 2 (0.33%)tests each. Figure 4 : Antimicrbial Sensitivity Testing f Quality Cntrl Strains & The Effect f Different Variables. Ttal ASTs n=600 Unaffected Tests n=578(96.4%) Variables affecting AST n = 22 (3.6%) Media n=5 (0.8%) Antimicrbial disk n=3 (0.5%) Inculums n=5 (0.8%) envirnmental factr=3 (0.5%) Zne f inhibitin incubatin n=2 (0.33%) Observatinal factr n=2 (0.33%) equipments n=2 (0.33%) :: 90 ::

3 GCSMC J Med Sci Vl (V) N (II) July-December 2016 Discussin: Antimicrbial susceptibility tests measure the ability f an antibitic r ther antimicrbial agent t inhibit bacterial grwth in vitr. This ability may be estimated (3) by either the dilutin methd r the diffusin methd. The dilutin methd: Fr quantitative estimates f antibitic activity, dilutins f the antibitic may be incrprated int brth r agar medium, which is then inculated with the test rganism. The lwest cncentratin that prevents grwth after vernight incubatin is knwn as the minimum inhibitry cncentratin (MIC) f the agent. The MIC value is then cmpared with knwn cncentratins f the drug btainable in the serum, and in ther bdy fluids t assess the likely clinical respnse. The Kirby-Bauer disc diffusin methd: Paper discs impregnated with a defined quantity f antimicrbial agent are placed n agar medium unifrmly seeded with the test rganism. A cncentratin gradient f the antibitic ccurs due t diffusin frm the disc and the grwth f the test rganism is inhibited at a particular distance frm the disc, that is related amng ther factrs t the susceptibility f the rganism. Agar: Mueller Hintn Agar is cnsidered as the best medium (4) fr the rutine susceptibility testing, since it has batcht-batch reprducibility, lw cncentratin f inhibitrs f sulphnamide, trimethprim and tetracyclines and prduces satisfactry results fr mst f the nnfastidius pathgens. Mueller-Hintn agar shuld be prepared frm a dehydrated base accrding t the manufacturer's recmmendatins. It is imprtant nt t verheat the medium. Cl the medium t C and pur int the plates. Allw t set n a level surface, t a depth f apprximately 4 mm. A 9 cm diameter plate requires apprximately 25 ml f the medium. When the agar has slidified, dry the plates fr immediate use fr minutes at 36 C by placing them in an upright psitin in the incubatr with the lids tilted. The medium shuld be such that cntrl zne sizes within the standard limits are prduced. Any unused plates may be stred in a plastic bag, which shuld be sealed and placed in the refrigeratr. Plates stred in this way can be kept fr 2 weeks. T ensure that the zne diameters are sufficiently reliable fr testing susceptibility t sulfnamides and c-trimxazle, Mueller-Hintn agar must have lw cncentratins f the inhibitrs thymidine and thymine. Each new lt f Mueller-Hintn agar shuld therefre be tested with a cntrl strain f Entercccus faecalis ( r 33l86) and a disc f c-trimxazle. A satisfactry lt f medium will give a distinct inhibitin zne f 20 mm r mre, that is essentially free f hazy grwth r fine clnies. Table 2 : Factrs affecting AST & their effect n Factr ph Misture it (variables). Variables If ph is lw:õ *Aminglycsides, Quinlnes & Macrlides lse ptency. * Tetracyclines have excess actin. Affects accuracy f susceptibility testing. Medium Excess f thymine reversibly inhibits cmpnent actin f antibitics like trimethprim grup. Thickness Antibitic discs: T minimize errr we shuld If thickness f medium is mre, grwth f rganism is less and vice versa. Use cmmercially available discs with the prper diameter (6 mm). Keep apprpriate distance between discs. Use disc with prper ptency. Stcks f antibitic discs shuld preferably be kept at - 20 C; the freezer cmpartment f a hme refrigeratr is cnvenient. A small wrking supply f discs can be kept in the refrigeratr fr up t 1 mnth. On remval frm the refrigeratr, the cntainers shuld be left at rm temperature fr abut l hur t allw the temperature t equilibrate. This prcedure reduces the amunt f :: 91 ::

4 Patel F V et al: Antimicrbial Susceptibility Tests cndensatin that ccurs when warm air reaches the cld cntainer. Antibitic discs shuld be placed within 15 minutes f swabbing. Inculum: Prepare the turbidity standard by puring 0.6 ml f a 1% (10 g/l) slutin f barium chlride int a l00-ml graduated cylinder, and filling t l00 ml with 1% (10 ml/l) sulfuric acid. The turbidity standard slutin shuld be placed in a tube identical t the ne used fr the brth sample. It can be stred in the dark at rm temperature fr 6 mnths, prvided it is sealed t prevent evapratin. Stcks f sterile cttn wl swabs n wden applicatr sticks shuld be prepared. These can be sterilized in tins, culture tubes, r n paper in the autclave. T prepare the inculum frm a primary culture plate, tuch the tps f each f the 3-5 clnies f similar appearance f the rganism t be tested, with a lp. When the inculum has t be made frm a pure culture, a lpful f the cnfluent grwth is similarly suspended in saline, r peptne water. Cmpare the tube with the turbidity standard and adjust the density f the test suspensin t that f the standard by either adding mre bacteria r mre sterile saline. Prper adjustment f the turbidity f the inculum is essential t ensure that the resulting lawn f grwth is cnfluent r almst cnfluent. Inculate the plates by dipping a sterile swab int the inculum. Remve excess inculum by pressing and rtating the swabs firmly against the side f the tube abve the level f the liquid. The mean MIC f the islates increases as the 3 inculum size prgressively increases frm 10 t 7 10 Clny Frming Units(CFUs). Identical results were nted when islates were maintained fr tw r fur days prir t testing. Inculum size shuld be carefully cntrlled when assessing the in vitr susceptibility. Size f zne inhibitin increases if inculum density is less. Size f zne inhibitin decreases if inculums is heavy. Incubatin: Knwledge f requirements fr incubatin is als needed as different rganisms have specific requirements, e.g., nn fastidius aerbic and facultative anaerbic bacteria require ambient air at 0 35 C fr generally hurs; while 24 hurs are needed fr S.aureus and Entercccus spp; and 5% CO 2 is needed fr Haemphilus spp. Equipment factrs: If there is prblem with petridish r measuring calibre, then als, the result f antimicrbial susceptibility test may be affected. Observatinal errr and ther envirnmental factrs als affect utcme f Antimicrbial susceptibility test. The final result f a disc diffusin test is influenced by a large number f variables. Sme f the factrs, such as the inculum density and the incubatin temperature, are easy t cntrl; but a labratry rarely knws the exact cmpsitin f a cmmercial medium, the antimicrbial cntent f the discs r the batch-t-batch variatins in their quality, and it cannt be taken fr granted. The results f the test must, therefre, be mnitred cnstantly by a quality cntrl prgram which shuld be cnsidered part f the prcedure itself. The precisin and accuracy f the test are cntrlled by the parallel use f a set f cntrl strains, with knwn susceptibility t the antimicrbial agents. These quality cntrl strains are tested using exactly the same prcedure as fr the test rganisms. The zne sizes shwn by the cntrl rganisms shuld fall within the range f diameters standardized. When results regularly fall utside this range, they shuld be regarded as evidence that a technical errr has been intrduced int the test, r that the reagents are at fault. Each reagent and each step in the test shuld then be investigated until the cause f the errr has been fund and eliminated. The quality cntrl prgram shuld use standard reference strains f bacteria that are tested in parallel with the clinical culture. They shuld preferably be run every week r with every fifth batch f tests, and in additin, every time that a new batch f Mueller Hintn agar r a new batch f discs is used. :: 92 ::

5 GCSMC J Med Sci Vl (V) N (II) July-December 2016 T avid errrs: Use antibitic discs f 6 mm diameter, with prper space between the discs kept in AST. Use crrect cntent f antimicrbial agent per disc. Stre supply f antimicrbial discs at -20 C. Use Mueller-Hintn medium fr antibitic sensitivity determinatin. Use apprpriate cntrl cultures. Use standard methdlgy fr the test. Use cded strains frm time t time fr internal quality cntrl. Everyne shuld be aware f the imprtance f QC. Training f persnnel with the crrect technique. Checking f expiry date f discs befre use. Maintaining f prper temperature f all the equipments required in AST. Peridic service and calibratin f instruments required in AST. Cnclusin: Internal quality assessment is a useful cmplementary apprach t external quality assessment and may detect prblem areas nt highlighted by ther cntrl methds. Educatin, accuracy and training is an imprtant part f the quality assurance prcess. Knwledge f atypical results fr different rganism agent cmbinatins may prvide warning f pssibly errneus results, and an understanding f the limitatins and surces f errr in disc diffusin methds cntributes significantly t the recgnitin, reslutin and avidance f errrs. References: 1. Labratry Methds and Strategies fr Antimicrbial Susceptibility Testing. (2007). In: Frbes B. A., Sahm D. F., Weissfeld A. C. (ed.) Bailey & Sctt's Diagnstic Micr bilgy. 12th ed. St. Luis, MO: Msby Elsevier; Waterwrth, P. M. (1951). A cmparative study f methds f testing sensitivity t antibitics and f the factrs influencing the results. Jurnal f Medical Labratry Technlgy 9, General principles f antimicrbial susceptibility testing; Basic Labratry Prcedures in Clinical Bacterilgy (WHO; 1991; 128 pages). 4. Kirby-Bauer Disk Diffusin Susceptibility Test Prtcl;Amarican sciety f micrbilgy; 01 April :: 93 ::