Below are some of the items that could possibly affect sample integrity and the result reported for a sample:

Transcription

1 Trubleshting Guide f PT Failures fr Micrbilgy Prgrams After lking at the Perfrmance Reprt fr failed results, it is imprtant t g ver the pssible errrs that culd have happened thrughut the testing prcess. This guide may help determine sample handling errrs when it cmes t testing Micrbilgy based PT samples. Belw are sme f the items that culd pssibly affect sample integrity and the result fr a sample: Cnditin f samples upn receipt What temperature did yu receive the samples at? Strage f Samples upn receipt What temperature did yu stre the samples at upn receipt? Handling instructins Did yu read the handling instructins fr the prgram befre testing the samples? Backgrund/Patient Histry Infrmatin n Wrksheets Is the rganism yu identified in the sample cnsidered a pathgen fr the sample surce and/r case histry infrmatin indicated n the Wrksheet? Sample Preparatin Was the sample equilibrated t a specific temperature befre testing? Was the sample recnstituted crrectly? Was the sample diluted crrectly? Media and Culture Cnditins Shuld yu have used primary culture media r selective culture media? Did yu read the sample surce and case histry infrmatin n the Wrksheet t determine what media and grwing cnditins t use? 1

2 Sample Handling Errrs If there was a prblem with the reprting ptin yu submitted fr a sample, n yur Perfrmance Reprt, please review the fllwing infrmatin fr prbable causes and/r slutins based n the type f testing. Acid Fast Stain Brightfield Micrscpy Participants are t submit a qualitative reprting ptin fr the quantity f Acid Fast Bacilli (AFB) using a Ziehl Neelsen stain and brightfield micrscpy. The reprting ptins are based n the WHO/IUATLD recmmended grading f sputum smear micrscpy results which invlve reading the slide with the 100x il immersin bjective. Results fr AFB using auramine stain with flurescent micrscpy cannt be at this time. If yu negative fr a sample that was psitive fr AFB, using Ziehl Neelsen stain If yu psitive fr a sample that was negative fr AFB, using Ziehl Neelsen stain Slide was wiped instead f bltted during the drying step Wrng side f the slide was stained Carbl fuchsin prepared frm pr quality reagents Carbl fuchsin reagent has expired r was stred in direct sunlight Carbl fuchsin staining time was less than 10 minutes Slide was ver declrized Check results fr the psitive cntrl rganism with the same lt f carbl fuchsin stain Declrizatin time was nt lng enugh Declrizing slutin has expired and culd be cntaminated Carbl fuchsin has dried n the smear Check results fr the negative cntrl rganism with the same lt f carbl fuchsin stain 2

3 Antimicrbial Susceptibility Testing Participants results fr antimicrbial susceptibility testing are evaluated based n the suggested antimicrbial grupings in the current versin f the CLSI dcument Perfrmance Standards fr Antimicrbial Susceptibility Testing: Infrmatinal Supplement, M100-SXX. Based n the rganism yu islated and the sample surce indicated n the Wrksheet, refer t the table in the CLSI dcument cntaining the suggested antimicrbial grupings fr fastidius rganisms r the table cntaining the suggested antimicrbial grupings fr nn-fastidius rganisms. Results fr ther antimicrbials nt listed in the CLSI dcument will be cnsidered unacceptable (UNACC). If the antimicrbial is n the list f CLSI acceptable antimicrbials, and Sensitive was instead f Resistant If the antimicrbial is n the list f CLSI acceptable antimicrbials, and Resistant was instead f Sensitive Check yur perfrmance reprt t determine if yu crrectly identified the rganism. The MIC interpretatins may be different depending n the rganism yu identified. Check the age f the inculum surce plate. Plate used t prepare inculum shuld be hurs. Check the cncentratin f rganism used t inculate the brth r agar MIC medium was at a high enugh cncentratin fr testing. Check t make sure the antimicrbial used fr testing is nt expired and the QC rganism tested with the same antimicrbial has a MIC within the acceptable range fr the agar r brth MIC assay. Check ph f the MIC media (ph shuld be 7.2 t 7.4) Ca++ and/r Mg++ cncentratin t high r t lw (fr Tetracyclines and Aminglycsides) Check perfrmance reprt t determine if yu crrectly identified the rganism. The MIC interpretatins may be different depending n the rganism yu identified. Check t make sure the cncentratin f rganism used t inculate the brth r agar MIC medium was nt abve the acceptable cncentratin fr testing. Check t make sure the antimicrbial used 3

4 fr testing is nt expired and the QC rganism tested with the same antimicrbial has a MIC within the acceptable range fr the agar r brth MIC assay. Check ph f the MIC media (ph shuld be 7.2 t 7.4) Ca++ and/r Mg++ cncentratin t high r t lw (fr Tetracyclines and Aminglycsides) Bacterial Culture and Identificatin The sample material sent ut fr the Bacterial Identificatin prgram will cnsist f aerbic, anaerbic r micraerphilic rganisms. Yu must review the case histry infrmatin and sample surce infrmatin fr each sample n yur Wrksheet in rder t determine the media and culture cnditins t use. Review yur recrds fr that sample, nting yur findings abut gram stain and mrphlgy (if perfrmed), mrphlgic characteristics n grwth media, results fr bichemical tests etc. If an rganism was and it was a negative sample species f rganism was Imprve ability t differentiate rganisms based n grwth patterns and bichemical tests Review yur recrds fr the sample, nting yur findings abut gram stain and mrphlgy (if perfrmed), mrphlgic characteristics n grwth media, results fr bichemical tests etc. If n grwth was and the sample cntained a pathgen The sample was stred at the wrng Check gram stain slide t see if rganism was missed Review Handling instructins t determine if sample was stred at the indicated and if sample was recnstituted crrectly. 4

5 Review Wrksheet fr sample surce and case histry infrmatin fr the sample. The PT prvider may have indicated fr yu t set up the culture t micraerphilic r anaerbic cnditins. If yu dn t d micraerphilic r anaerbic cultures at yur lcatin, submit a reprting ptin that indicates yu dn t d this type f testing at yur lcatin. Failure t d s will result in an UNACC fr that sample n yur perfrmance reprt. Bld Parasites The sample material sent ut fr the Bld Parasite prgram r Malaria prgram cnsist f Giemsa stained thick r thin bld smears. The 2 sample frmat Bld Parasites prgram (BLPA432) nly cnsists f Giemsa stained thin bld smears. species f parasite was If parasite(s) were and the sample was negative fr parasites If n parasite(s) were and the sample was psitive fr parasite(s) Imprve ability t differentiate parasites at different stages f grwth. Cmpare parasite mrphlgy with thse described in a reference textbk. If unsure f the species, nly reprt t the genus level. Failure t d s might result in an unacceptable grade. In thick films, rganisms tend t be mre cmpact and denser than in thin films, which might affect identificatin. The wrng slide was reviewed Identified an element as a parasite Review the slide again and refer t reference textbks t review the mrphlgy f the parasite yu thught yu bserved n the slide. The bld film wasn t scanned n a lw pwer bjective befre ging t a high pwer bjective. First screen the smear at a lw magnificatin (10 r 20 bjective), t detect large parasites (micrfilaria) then examine the 5

6 smear using il immersin bjective. CLSI recmmend scanning at least 300 il immersin fields fr the determinatin f N Parasite Seen. The sample may have cntained a lw cncentratin f parasite. Dermatphyte Testing The DERS435 prgram is designed fr participants t identify the presence/absence f dermatphytes n Dermatphyte Test Medium (DTM). Any ther type f media is nt suitable fr this prgram. In additin t bserving fr a red clr change, the interpretatin f DTM grwth shuld include a review f the grss and micrscpic clny mrphlgy. Dermatphytes grwn n DTM are usually lightly pigmented (white, tan, r cinnamn-clred), whereas cmmn saprphytes are ften darkly pigmented. Sme rganisms will grw n DTM after 48 hurs but will change the DTM t a yellw clr, which des nt indicate the presence f a dermatphyte. If psitive was negative fr dermatphytes If negative was psitive fr dermatphytes The sample was recnstituted incrrectly There may be a prblem with the lt f DTM used fr culture Review yur recrds t cnfirm grwth f the QC rganism was acceptable n the lt f DTM yu used fr testing. The culture cnditins were nt suitable fr grwth There may have been a lw cncentratin f dermatphyte in the sample 6

7 Genital Culture The sample material fr the Genital Culture prgram cnsists f nrmal flra and/r pathgens islated frm the genital tract. Review the sample surce infrmatin n yur Wrksheet t determine which media and culture cnditins t use. If grwth was negative fr rganisms species f rganism was Imprve ability t differentiate rganisms based n grwth patterns and bichemical tests. Review yur recrds fr the sample, nting yur findings abut gram stain and mrphlgy (if perfrmed), mrphlgic characteristics n grwth media, results fr bichemical tests etc. If unsure f the species, nly reprt t the genus level. Failure t d s might result in an unacceptable grade. If n grwth was psitive fr a pathgen The sample was incrrectly recnstituted Review Handling instructins t determine if sample was stred at crrect temperature upn receipt and if sample was recnstituted crrectly. Review Wrksheet fr sample surce infrmatin. Based n the sample surce, determine if crrect media and incubatin was used fr culture. 7

8 Gram Stain and Mrphlgy The sample material fr the Gram Stain prgram cnsist f unstained heat fixed smears. If gram negative micrrganism was and the sample cntained a gram psitive micrrganism If gram psitive micrrganism was and the sample cntained a gram negative micrrganism If n micrrganisms bserved was and the sample cntained a gram negative micrrganism and/r gram psitive micrrganism The slide was ver-declrized Failure t include the mrdanting (idine) step The slide was under-declrized The declrizatin step was t shrt r was mitted The gram stain dried n the smear The slide was wiped instead f bltted during the drying step The wrng side f the slide was stained The slide wasn t reviewed n lw pwer (10x bjective). Lk at many micrscpic fields and in different areas f the slide. Mld Culture If grwth was and there was n rganism present in the specimen species f rganism was Yu need t imprve yur ability t differentiate rganisms based n grwth patterns and micrscpic features. Macrscpic examinatin Clnial mrphlgy Surface pigment Reverse pigment Grwth n cyclheximide cntaining agar 8

9 Micrscpic examinatin tease munt r sctch tape preparatin using Lactphenl Blue (LPCB) Under light micrscpe, examine the slide fr the presence, shape, size and attachment f cnidia. Cmpare features with thse described in a reference textbk If n grwth was psitive fr a pathgen Review Handling instructins t determine if sample was stred at crrect temperature upn receipt and if sample was recnstituted crrectly. Review the lactphenl cttn blue stained smear (if used at yur lcatin) t see if the rganism was missed n the slide Review the Wrksheet fr the sample surce and case histry infrmatin fr the sample. Based n the case histry and sample surce infrmatin, determine if the crrect media and incubatin cnditins were used fr culturing the sample. Neisseria gnrrheae Screening If psitive fr N. gnrrheae was, and the sample was negative fr N. gnrrheae If negative fr N. gnrrheae r n grwth was, and the sample was psitive fr N. gnrrheae The sample was recnstituted incrrectly The chclate agar r selective media, such as mdified Thayer-Martin, Martin Lewis r New Yrk City medium, wasn t equilibrated t rm temperature befre inculating with the sample. The inculated plate was nt incubated in a 9

10 carbn dixide atmsphere. Check yur QC recrds fr the lt # f media and QC rganism used t determine if there was a prblem with the levels f CO2. PVA Smear The sample material fr the PVA Smear prgram cnsists f mercury fixed PVA fecal smears. Refer t the case histry infrmatin n the Wrksheet fr details regarding the histry f the patient. species f parasite was If a parasite was negative fr parasites If n parasites were psitive fr parasites Need t imprve ability t differentiate parasites at the different stages f grwth. Cmpare the parasite mrphlgy with thse described in a reference textbk. If unsure f the species, nly reprt t the genus level. Failure t d s might result in an unacceptable grade. There may be an artifact in the sample. Artifacts shuld be cnsidered n the basis f size, shape, lack f rganelles and defining feature, and variable reactivity with cmmn stains Cmpare the mrphlgy with thse described in a reference textbk The entire cverslip wasn t scanned n the 10x bjective at lw light befre ging t the high pwer bjective. If yu see smething suspicius n the lw pwer bjective, g t the 40x bjective t see mre detailed mrphlgy f the element in questin. High dry pwer examinatin shuld include at least ne third f the smear area. 10

11 Streptcccus A Screening If psitive fr Grup A Streptcccus was, negative fr Grup A Streptcccus If negative fr Grup A Streptcccus was, psitive fr Grup A Streptcccus The sample was recnstituted incrrectly The lt number f selective media used may have expired The culture cnditins were nt suitable fr grwth Urine Clny Cunt If a result abve r belw the acceptable value was If grwth was and the sample was negative fr rganisms If n grwth was fr a psitive sample Review the handling instructins fr the prgram, t make sure yu recnstituted and/r diluted the sample prperly. Make sure yu yur results in the same units as the reprting ptins in OASYS. The reprting ptins fr urine clny cunt n the OASYS website are in clnies/ml. Check grwth f the QC rganism n the lt number f media yu used The lt f media used may have expired The culture cnditins were nt suitable fr grwth The sample was recnstituted incrrectly Review Handling instructins 11

12 Urine Culture If grwth was and the sample was negative fr rganisms species f rganism was If n grwth was fr a psitive sample Imprve ability t differentiate rganisms based n grwth patterns and bichemical tests Review yur recrds fr the sample, nting yur findings abut gram stain and mrphlgy (if perfrmed), mrphlgic characteristics n grwth media, results fr bichemical tests etc. The culture cnditins were nt suitable fr grwth The sample was recnstituted incrrectly Review Handling instructins Wet Munt The sample material fr the Wet Munt prgram cnsists f frmalin preserved fecal suspensin. Refer t the case histry infrmatin n the Wrksheet fr details regarding the histry f the patient. species f parasite was If parasite(s) was negative fr parasites Imprve ability t differentiate parasites at different stages f grwth. Cmpare the parasite mrphlgy with thse described in a reference textbk. If unsure f the species, nly reprt t the genus level. Failure t d s might result in an unacceptable grade. There may be an artifact in the sample. Artifacts shuld be cnsidered n the basis f size, shape, lack f rganelles and defining feature Cmpare the mrphlgy with thse described in a reference textbk 12

13 If n parasite(s) was psitive fr parasites The sample was stred at the wrng The wrng sample was tested The entire cverslip wasn t scanned n the 10x bjective at lw light befre ging t the high pwer bjective. If yu see smething suspicius n the lw pwer bjective, g t the 40x bjective t see mre detailed mrphlgy f the element in questin. High dry pwer examinatin shuld include at least ne third f the cverslip area. Yeast Culture If grwth was fr a negative sample species f rganism was If n grwth was fr a psitive sample Imprve ability t differentiate rganisms based n grwth patterns n selective media, results frm germ tube test, urease test and ther bichemical tests. Review Handling instructins t determine if sample was stred at crrect temperature upn receipt and if sample was recnstituted crrectly. Review gram stain slide (if perfrmed) t see if the rganism was missed Review Wrksheet fr sample surce and case histry infrmatin. Based n case histry and sample surce infrmatin, determine if the crrect media and incubatin cnditins were used fr culture. 13