STANDARD DIAGNOSTIC METHODSSTANDARD DIAGNOSTIC METHODS

OPTIMAL USE AND OPTIMAL USE AND ALGORITHMSALGORITHMS

THE ENEMYTHE ENEMY………………..

1/25,000th OF AN INCH

ROLE(S) OF THE ROLE(S) OF THE LABORATORYLABORATORY…………………………

Primary Care Primary Care –– for better patient for better patient management management

Public Health Public Health –– for appropriate for appropriate implementation of infection control implementation of infection control and public health measures to control and public health measures to control the transmission of tuberculosisthe transmission of tuberculosis

THE EXPECTATIONS (CDC)THE EXPECTATIONS (CDC)Specimens reach the laboratory within 24 h of Specimens reach the laboratory within 24 h of collection.collection.Physician receive AFB stain results within 24 Physician receive AFB stain results within 24 h of laboratoryh of laboratory’’s receipt of specimen.s receipt of specimen.NAAT results NAAT results ≤≤ 48 h of specimen collection48 h of specimen collectionPositive cultures detected within 14 days of Positive cultures detected within 14 days of specimen collection.specimen collection.MTB is identified MTB is identified ≤≤ 21 days of specimen 21 days of specimen receipt.receipt.

NONTUBERCULOUS NONTUBERCULOUS MYCOBACTERIA (NTM)MYCOBACTERIA (NTM)

IncreasingIncreasing incidence of infections incidence of infections caused by these organismscaused by these organismsIncreasingIncreasing number of speciesnumber of species>> 1975: ~ 30 species1975: ~ 30 species>> 30 years later: >120 species30 years later: >120 species

HIGHLIGHT IMPORTANT ASPECTSREGARDING THE LABORATORY DIAGNOSIS OF MYCOBACTERIAL INFECTIONS

SafetySafetyLevels of Laboratory and Levels of Laboratory and Referral ServicesReferral ServicesClinical Significance of Clinical Significance of MycobacteriumMycobacterium spp.spp.Specimen Types, Specimen Types, Collection, Transport and Collection, Transport and StorageStorageDetection of Detection of MycobacteriaMycobacteriaIdentification ProceduresIdentification Procedures

M48-A

SAFETY SAFETY –– RISK ASSESSMENTRISK ASSESSMENTUsed to determine the type of practices to Used to determine the type of practices to employ in the laboratoryemploy in the laboratoryWhoWhoFactors to take into accountFactors to take into account>> Level of TB diagnostic services offeredLevel of TB diagnostic services offered>> Laboratory designLaboratory design>> TB prevalenceTB prevalence>> Rate of multiRate of multi--drug resistant MTBdrug resistant MTB>> Whether or not aerosolWhether or not aerosol--generating procedures generating procedures

are performed as well as their respective are performed as well as their respective frequencyfrequency

CLINICAL SIGNIFICANCE OF CLINICAL SIGNIFICANCE OF MYCOBACTERIUMMYCOBACTERIUM SPP.SPP.

M. tuberculosisM. tuberculosis complexcomplex>>Most clinically significant, therefore its Most clinically significant, therefore its

detection should be a primary focus of detection should be a primary focus of the laboratorythe laboratory

>>NOT in the environmentNOT in the environment>> Isolation almost always signifies diseaseIsolation almost always signifies disease

CLINICAL SIGNIFICANCECLINICAL SIGNIFICANCE

NTM NTM -- WHY MORE ATTENTION?WHY MORE ATTENTION?>> Associated with AIDS Associated with AIDS >> Recognition that NTM lung disease is Recognition that NTM lung disease is

encountered with increasing frequency in encountered with increasing frequency in the nonthe non--AIDS populationAIDS population

>> Improvement in Improvement in mycobacteriologymycobacteriologylaboratory methodslaboratory methods»» Enhanced isolationEnhanced isolation»» More rapid and accurate identification of More rapid and accurate identification of NTMsNTMs

NTMsNTMsDetermining clinical significance Determining clinical significance when an NTM is isolated:when an NTM is isolated:>> Clinical setting and hostClinical setting and host>>Organism species and its pathogenic Organism species and its pathogenic

potentialpotential>> Source of the culture isolate & likelihood Source of the culture isolate & likelihood

for contaminationfor contamination>>Quantification of organisms detected Quantification of organisms detected

both in culture and AFB smearsboth in culture and AFB smears>>Number of positive culturesNumber of positive cultures

CLINICAL SIGNIFICANCE CLINICAL SIGNIFICANCE -- NTM: NTM: LABORATORY INDICATORSLABORATORY INDICATORS

Bacterial burdenBacterial burden>> Reflected by number of organisms seen in AFB Reflected by number of organisms seen in AFB

smearssmears>> CultureCulture

»» More sensitive than smear so will detect smaller More sensitive than smear so will detect smaller numbers of organismsnumbers of organisms

»» ATS recommendation = ATS recommendation = quantitatequantitate mycobacterial mycobacterial growth on solid media culturesgrowth on solid media cultures

Number of positive culturesNumber of positive cultures>> Indicative of disease relevanceIndicative of disease relevance>> ATS recommendation = 3 sputum specimens to ATS recommendation = 3 sputum specimens to

screen for NTM diseasescreen for NTM disease

CLINICAL SIGNIFICANCE CLINICAL SIGNIFICANCE -- NTM: NTM: LABORATORY INDICATORSLABORATORY INDICATORS

Microbiologic criteria for diagnosing NTM Microbiologic criteria for diagnosing NTM lunglung diseasedisease>> Positive culture results from at least 2 Positive culture results from at least 2

expectorated sputumexpectorated sputum samplessamples>> Positive culture result from at least Positive culture result from at least 1 BW or BAL1 BW or BAL>> TransbronchialTransbronchial or other lung biopsy with or other lung biopsy with

mycobacterial histopathologic features and 1 mycobacterial histopathologic features and 1 positive culture positive culture OROR biopsy showing biopsy showing mycobacterial histopathologic features and 1 or mycobacterial histopathologic features and 1 or more sputum or BWs that are culturemore sputum or BWs that are culture--positive for positive for NTMNTM

Mixed culturesMixed cultures

OR

OR

An Official ATS/IDSA Statement: Diagnosis, Treatment,And Prevention of Nontuberculous Mycobacterial Diseases. 2007. Am. J. Respir. Crit. Care Med. 175: 367-416.

SPECIMEN TYPES, SPECIMEN TYPES, COLLECTION, TRANSPORT, COLLECTION, TRANSPORT, AND STORAGEAND STORAGE

SPECIMENSPECIMENQUALITYQUALITY

KEY POINTS TO ENSURE KEY POINTS TO ENSURE SPECIMENT QUALITYSPECIMENT QUALITY

PROPER SPECIMEN COLLECTION.PROPER SPECIMEN COLLECTION.PROPER SPECIMEN COLLECTION.PROPER SPECIMEN COLLECTION.PROPER SPECIMEN COLLECTION.PROPER SPECIMEN COLLECTION.

KEY POINTS TO ENSURE KEY POINTS TO ENSURE SPECIMENT QUALITY (conSPECIMENT QUALITY (con’’t)t)

Avoid swabsAvoid swabsRefrigerate specimens until processed Refrigerate specimens until processed (except for blood specimens)(except for blood specimens)Reject the following specimensReject the following specimens>> Dry swabsDry swabs>> Gastric washing Gastric washing notnot neutralized and > 1 h oldneutralized and > 1 h old

SPECIMEN PROCESSINGSPECIMEN PROCESSING

SPECIMEN SPECIMEN DECONTAMINATIONDECONTAMINATION

Viable AFBDead bacteria

SPECIMEN PROCESSINGSPECIMEN PROCESSINGContamination ratesContamination rates>> Expected: 2 to 5%Expected: 2 to 5%

»» If <2%, processing method may be killing many AFBIf <2%, processing method may be killing many AFB»» If >5%, processing may be inadequate.If >5%, processing may be inadequate.

>> Useful to monitor the recovery rate of Useful to monitor the recovery rate of M. M. gordonaegordonae

Digestion/ decontamination methods Digestion/ decontamination methods >> No clear consensusNo clear consensus>> Number of methods Number of methods –– appendicesappendices>> NTMsNTMs and cystic fibrosis patients and cystic fibrosis patients –– specific specific

decontamination methods employeddecontamination methods employed»» TwoTwo--step NALCstep NALC--NaOHNaOH--oxalic methodoxalic method»» ChlorhexidineChlorhexidine methodmethod

DECONTAMINATING AGENTSDECONTAMINATING AGENTSDecontaminating reagents Decontaminating reagents -- examplesexamples>> NN--acetylacetyl--LL--cysteine (NALC) with NaOH cysteine (NALC) with NaOH

(commonly used in US)(commonly used in US)>> Sodium lauryl sulfate (commonly used in Sodium lauryl sulfate (commonly used in

EuropeEurope))>> Whole host of others: e.g. zephiranWhole host of others: e.g. zephiran--trisodium trisodium

phosphatephosphateAfter decontamination, adding buffered After decontamination, adding buffered solution helps not only to neutralize the solution helps not only to neutralize the base but alsobase but also>> Dilutes toxic substancesDilutes toxic substances>> Decrease specific gravity so that AFB can be Decrease specific gravity so that AFB can be

readily concentrated by centrifugationreadily concentrated by centrifugation

SPECIMEN PROCESSING SPECIMEN PROCESSING ((concon’’tt))

ConcentrationConcentration>> RCF = 1.12 RCF = 1.12 RmaxRmax (rpm/1000)(rpm/1000)22

>> AFB smear and culture sensitivity depend AFB smear and culture sensitivity depend on optimum RCFon optimum RCF

>>Minimum recommended RCF = 3000 Minimum recommended RCF = 3000 x gx gfor 15 minutesfor 15 minutes

Measures to prevent crossMeasures to prevent cross--contamination of specimenscontamination of specimens

DETECTION OF DETECTION OF MYCOBACTERIAMYCOBACTERIA

MicroscopyMicroscopyNucleic acid amplification tests for Nucleic acid amplification tests for direct detection of direct detection of M. tuberculosisM. tuberculosis in in clinical specimensclinical specimens

SENSITIVITY AND SPECIFICITY SENSITIVITY AND SPECIFICITY OF AFB SPUTUM SMEARSOF AFB SPUTUM SMEARS

REFERENCE SENSITIVITY (%) SPECIFICITY (%)Alluoch etal 39 99.8

Boyd, Marr etal 22 99.3

Burdash etal 43 99.9

Levy etal 53 99.8

Lipsky etal 33 99.8

Strumpf etal 78 99.7

FACTORS INFLUENCING FACTORS INFLUENCING AFB SMEARAFB SMEARSENSITIVITY:SENSITIVITY: Specimen type, species of infecting Specimen type, species of infecting mycobacteriamycobacteria, efficiency of decontamination & , efficiency of decontamination & concentration procedures, smear thickness, type of concentration procedures, smear thickness, type of stain, extent of decolorization or the type of stain, extent of decolorization or the type of counterstain used, training & experience of staffcounterstain used, training & experience of staffSPECIFICITY:SPECIFICITY: not using of not using of ‘‘bulkbulk’’ staining staining trays/containers, experience of trays/containers, experience of microscopistmicroscopistreagents for performing the stain (as well as all reagents for performing the stain (as well as all other components of digestion) are not properly other components of digestion) are not properly filtered or replaced. Common sources of AFB:filtered or replaced. Common sources of AFB: tap tap water, infrequently cleaned distilled water reservoirs.water, infrequently cleaned distilled water reservoirs.

MISCELLANEOUSMISCELLANEOUSUse of the fluorochrome stain strongly Use of the fluorochrome stain strongly recommended because of simplicity, recommended because of simplicity, sensitivity & speed.sensitivity & speed.False + stains should occur at a rate of False + stains should occur at a rate of <1% of all stained specimens.<1% of all stained specimens.What about screening for quality of What about screening for quality of sputum? NOsputum? NO> Lower respiratory tract secretions preferred> BUT, specimens with significant oropharyngeal

secretions can still grow MTB

EXAMINATION & EXAMINATION & REPORTINGREPORTING

Smear size = 1 x 2 cmSmear size = 1 x 2 cmExamine the equivalent of 300 OIFsExamine the equivalent of 300 OIFsSmear results must be quantified to be Smear results must be quantified to be meaningfulmeaningful……important that important that magnification used be taken into magnification used be taken into consideration so that results are consideration so that results are equivalent to 300 OIFs seen under equivalent to 300 OIFs seen under fuchsin stains examined at 1000X.fuchsin stains examined at 1000X.

DETECTION OF AFB BY CULTUREDETECTION OF AFB BY CULTUREKey to successful culturesKey to successful cultures>> Combination of different culture media is required to Combination of different culture media is required to

optimize the sensitivity of culture.optimize the sensitivity of culture.»» For example: an agarFor example: an agar--based or eggbased or egg--based medium, selective or based medium, selective or

plain plain ANDAND a liquid medium.a liquid medium.>> Depending on whether suspecting a particular AFB Depending on whether suspecting a particular AFB

infection, duplicate cultures on primary media should be infection, duplicate cultures on primary media should be inoculated at 30inoculated at 30--33 degrees C.33 degrees C.

>> Certain mycobacterial species have special nutritional Certain mycobacterial species have special nutritional requirements: requirements: M. M. genavensegenavense, M. , M. haemophilumhaemophilum and and M. M. paratuberculosisparatuberculosis

Advantages and disadvantages of media agarAdvantages and disadvantages of media agar--, egg, egg--based and liquid mediabased and liquid media

AFB CULTURE (AFB CULTURE (concon’’tt))Media SelectionMedia Selection>> Liquid Liquid vsvs solid mediasolid media>>Depends on the type of clinical specimenDepends on the type of clinical specimen»» Specimens from patients on antiSpecimens from patients on anti--TB therapyTB therapy»» Extrapulmonary specimens Extrapulmonary specimens –– inoculate in inoculate in

duplicate (one w/ and one w/o antibiotics) due to duplicate (one w/ and one w/o antibiotics) due to generally low numbers of organisms presentgenerally low numbers of organisms present»» Skin, bone and joints: inoculate to media Skin, bone and joints: inoculate to media

supplemented with an additional iron sourcesupplemented with an additional iron sourceHeminHeminFerric ammonium citrateFerric ammonium citrateCommercial XCommercial X--stripsstrips

AFB CULTURE (AFB CULTURE (concon’’tt))Examination of growthExamination of growth>> Solid media Solid media

»» Necessary to incubate a minimum of 4 weeksNecessary to incubate a minimum of 4 weeks»» CDCCDC--recommended criteria for colony counts:recommended criteria for colony counts:

>> Liquid mediaLiquid media»» Confirmation for presence of AFBConfirmation for presence of AFB»» Visual checksVisual checks

AFB CULTURE (AFB CULTURE (concon’’tt))

ContaminationContamination>> Sources: specimen, contaminated reagents Sources: specimen, contaminated reagents

or the environmentor the environment>>Negative control for each batch of Negative control for each batch of

specimens processed = sterile water or specimens processed = sterile water or buffer buffer »» If + for AFB, results of all processed specimens If + for AFB, results of all processed specimens

in the batch become invalidin the batch become invalid»» Recommendations for bacterial contaminationRecommendations for bacterial contamination

RECOMMENDATIONS:

AFB CULTURE (AFB CULTURE (concon’’tt))ContaminationContamination>> CrossCross--contaminationcontamination»» IndicationsIndications»» Investigate episodes thoroughly and take Investigate episodes thoroughly and take

corrective measures:corrective measures:

AFB CULTURE (AFB CULTURE (concon’’tt))

Recommended that important clinical Recommended that important clinical isolates be frozen at isolates be frozen at ––70 degrees C and 70 degrees C and saved for a minimum of 1 yearsaved for a minimum of 1 year>> Prepare suspensions in water, saline or 7H9 Prepare suspensions in water, saline or 7H9

broth; homogenize, and adjust to McFarland No. broth; homogenize, and adjust to McFarland No. 1 turbidity standard1 turbidity standard

>> Positive cultures in liquid media may be stored Positive cultures in liquid media may be stored directly in small aliquots by freezing at directly in small aliquots by freezing at ––70 70 degrees Cdegrees C

ReportingReporting

AFB CULTURE (AFB CULTURE (concon’’tt))

Quality ControlQuality Control>>MediaMedia: recommendations for prepared : recommendations for prepared

media including QC organismsmedia including QC organisms>> Specimen Processing and Isolation of Specimen Processing and Isolation of

MycobacteriaMycobacteria»» Analyze data for 3Analyze data for 3--6 months to determine an 6 months to determine an

overall average or a normal trend for the overall average or a normal trend for the individual laboratoryindividual laboratory»» Once parameters established, should remain Once parameters established, should remain

uniform with minor fluctuationsuniform with minor fluctuations

PARAMETERSPARAMETERSTotal specimens processedTotal specimens processedTotal and % AFB smearTotal and % AFB smear--positive and smearpositive and smear--negativenegativeTotal and % AFB cultureTotal and % AFB culture--positive from smearpositive from smear--negative and smearnegative and smear--positive specimenspositive specimensTotal and % cultures positive for MTB and Total and % cultures positive for MTB and NTMsNTMsAverage time to detection of AFBAverage time to detection of AFB--positive cultures positive cultures (smear(smear--negative and smearnegative and smear--positive), TB and NTMpositive), TB and NTMBacterial contamination rateBacterial contamination rateRecords of processing: technologist , all specimens Records of processing: technologist , all specimens in batch, negative control results, incubation in batch, negative control results, incubation temperaturetemperature

IDENTIFICATIONIDENTIFICATION

Differentiation of Differentiation of M. tuberculosisM. tuberculosis from otherfrom othermycobacteria represents an important publicmycobacteria represents an important publichealth issue.health issue.

IDENTIFICATIONIDENTIFICATIONPhenotypicPhenotypic: : >> Conventional biochemical tests Conventional biochemical tests >>HPLCHPLC

GenotypicGenotypic>>Hybridization assaysHybridization assays>> Amplification followed by sequencing or Amplification followed by sequencing or

REAREA

“TB or not TB, that is congestion. Consumption be done about it? Of cough, of cough. But it takes a lung, lung time.”