The Laboratory s Role in TB Diagnosis and Treatment

Transcription

1 TB Nurse Case Management San Antonio, Texas December 8-10, 2009 The Laboratory s Role in TB Diagnosis and Treatment EDWARD BANNISTER, Ph.D. December 9, 2009 The Laboratory s Role in TB Diagnosis and Treatment EDWARD R. BANNISTER, Ph.D. ASSISTANT DIRECTOR LABORATORY SERVICES SECTION DALLAS COUNTY DEPARTMENT OF HEALTH AND HUMAN SERVICES 1

2 INTRODUCTION MANY SPECIES ARE PROMINENT PATHOGENS M. tuberculosis complex M. leprae Nontuberculosis Mycobacteria (NTM) or atypical mycobacteria MICROBIOLOGICAL ASPECTS ONLY GENUS IN THIS FAMILY AEROBIC GROW BEST IN INCREASED CARBON DIOXIDE CELL WALL HIGH IN LIPID CONTENT SPECIAL STAINING NECESSARY TO VISUALIZE SLOW GROWERS (>7 DAYS) VS. RAPID GROWERS (<7 DAYS) 2

3 MICROBIOLGICAL ASPECTS CONT D COLONIES CAN BE PIGMENTED OR NONPIGMENTED ON EXPOSURE TO LIGHT MOST SPECIES DO NOT REQUIRE COMPLEX GROWTH MEDIA M. leprae HAS NEVER BEEN GROWN IN LABORATORY (OUTSIDE OF LIVING CELLS) CONT D MYCOBACTERIA CAN SURVIVE ON INANIMATE OBJECTS IF PROTECTED FROM SUNLIGHT FOR WEEKS TO MONTHS. M. tuberculosis CAN SURVIVE SEVERAL MONTHS ON SURFACES OR IN SOIL OR COW DUNG IF ANIMALS WERE INFECTED TEMPERATURE > 65 C FOR 30 MINUTES AND UV LIGHT SUN EASILY KILLS RESISTANT TO SEVERAL ACIDS AND ALKALIS THAN OTHER NON-SPORE FORMING BACTERIA 3

4 HABITATS OBLIGATE PATHOGEN M. tuberculosis AND M. leprae REQUIRE LIVING TISSUE TO REPLICATE WHETHER HUMAN OR ANIMAL. NTM FREE LIVING WATER, LAKES, RIVERS, WET-SAND. 1. TAP WATER MANY SPECIES CAN BE FOUND HERE 2. NOT NORMAL BACTERIAL FLORA OF HUMANS OR ANIMALS; MAY BE ISOLATED FROM SKIN, UPPER RESPIRATORY TRACT 3. CLINICAL SIGNIFICANCE BECOMES QUESTIONABLE CERTAIN SITUATIONS SPECIMEN COLLECTION MAJORITY ARE FROM RESPIRATORY TRACT ( SPUTUM, TRACHEAL, BRONCHIAL ASPIRATE, BAL). OTHERS URINE, GASTRIC ASPIRATE(CHILDREN) CSF, AND TISSUES BLOOD, FECAL MOSTLY IMMUNOCOMPROMISED PATIENTS 4

5 SPECIMEN CONTAINERS STERILE, LEAKPROOF AND DISPOSABLE, NO FIXATIVE NO WAXED CONTAINERS NO SWABS IF TRANSPORT DELAYED > 1 HOUR, REFRIGERATE LABORATORY SAFETY CLASS I OR CLASS II BIOLOGICAL SAFETY CABINET BEST IF AREA IS UNDER NEGATIVE AIR PRESSURE AND HOODS ARE VENTED TO TOP OF BUILDING PERSONAL PROTECTIVE EQUIPMENT MUST BE WORN MASKS, GLOVES AND ETC. IF SHIPPING ORGANISM TO ANOTHER LABORATRY YOU MUST FOLLOW IATA GUIDELINES AND SEND AS AN INFECTIOUS AGENT. INDIVIDUAL MUST BE CERTIFIED FOR THIS PROCEDURE. 5

6 SPUTUM TYPE 1. EXPECTORATED OR INDUCED 2. EARLY MORNING 3. THREE ON CONSECUTIVE DAYS 4. NO POOLING INITIAL EVALUATION TWO SMEAR POSITIVE SUFFICIENT FOR DIAGNOSIS THREE, HOWEVER STILL RECOMMENDED CULTURE FOLLOW-UP CONSIDER TO DETERMINE EFFECTIVENESS OF THERAPY OTHER SPECIMEN TYPES BRONCHIAL ASPIRATE, BAL, FINE NEEDLE ASPIRATE, BIOPSY GASTRIC LAVAGE FLUIDS BEST FOR CHILDREN URINE, BODY FLUIDS, TISSUES, ABSCESSES BLOOD BLOOD CULTURE BOTTLE FECAL (MAC) 6

7 UNACCEPTABLE SPECIMENS AMOUNT TOO SMALL - > 5 ML PREFERED LOOKS LIKE SALIVA DRIED SWABS POOLED SPUTUM OR URINE BROKEN SAMPLE CONTAINERS > 7 DAYS FROM COLLECTION TO DELIVERY TO LAB SUMMARY SPUTUM YOU ARE THE MOST IMPORTANT ASPECT IN THIS PROCESS > 5 ML BEST ALWAYS 3 ON THREE CONSECUTIVE DAYS NO POOLING DELIVER TO LAB WITHIN ONE HOUR OR REFRIGERATE STERILE CONTAINERS, TIGHT FITTING LID LABEL, LABEL, LABEL 7

8 SPECIMEN PROCESSING DECONTAMINATION AND DIGESTION 1. NORMALLY STERILE SPECIMENS NO DECONTAMINATION NECESSARY CONCENTRATION BY CENTRIFUGATION MAY HELP 2. CONTAMINATED SPECIMENS - TREAT TO REDUCE OR ELIMINATE OTHER BACTERIA THAT CAN OVERGROW THE MYCOBACTERIA AS THESE ARE SLOW GROWERS 3. METHODS SEVERAL DIFFERENT METHODS ARE AVAILABLE AND ALL ARE SATISFACTORY. NaLC-NaOH NaOH MOST IN US. LABORATORIES USUALLY CALCULATE CONTAMINATION RATE AND SHOULD BE NO MORE THAN 5%. CONCENTRATION SPECIMENS ARE CENTRIFUGED TO CONCENTRATE SPECIAL CENTRIFUGES ARE USED SO IN CASE TUBE BREAKS NO AEROSOL WOULD BE CREATED AND CONTAMINATE THE ROOM 8

9 STAINING MOST RAPID AND INEXPENSIVE WAY TO DIAGNOSE TUBERCULOSIS RAPID MEANS TO DIAGNOSE MOST CONTAGIOUS PATIENTS TWO WAYS - ACID-FAST CONVENTIONAL ZIEHL- NEELSEN, KINYON -FLUORCHROME NOT ALL NTM WILL STAIN, STILL MOST SENSITIVE - OTHER ORGANISMS CAN STAIN WITH THE ACID-FAST STAIN SUCH AS NOCARDIA, RHODOCOCCUS, CRYPTOSPORIDIUM - CANNOT DISTINGUISH BETWEEN MTB AND NTM, BUT MORPHOLOGY MAY HELP STAINING GENERAL COMMENTS SENSITIVITY IN COMPARISON TO CULTURE RANGES FROM 22 78% - DEPENDS ON SPECIMEN AND LABORATORY ESTIMATED DETECTION LEVELS BY SMEAR ARE 5-10,000/ML OF SPUTUM CULTURE VIABLE ORGANISMS 9

10 REPORTING MANY WAYS NO ACID-FAST BACILLI SEEN DCHHS < 1/FIELD 1-10/FIELD10/FIELD > 10/FIELD OTHERS 1+ TO 4+ CULTURE PROCEDUES MEDIA USED SOLID LIQUID SOLID TYPES LOWENSTEIN-JENSEN (LJ) MIDDLEBOORK 7H10 MIDDLEBROOK 7H11 SELECTIVE GRUFT MODIFICATION OF LJ MIDDLEBROOK 7H10 AND 11 WITH ANTIBIOTICS 10

11 LIQUID BACTEC RADIOMETRIC SYSTEM CO2 BACTEC MGIT FLUORESCENCE BAC/T/ALERT CO2 ISOLATOR TUBE BLOOD MIDDLEBROOK 7H9 MB REDOX ESP CULTURE SYSTEM INCUBATION TIME NORMAL 6-8 WEEKS BEFORE REPORTING NEGATIVE IF SMEAR POSITIVE USUALLY WILL BE CULTURE POSITIVE IN 2 3 WEEKS DEPENDING ON THE NUMBER OF ORGANISMS 11

12 IDENTIFICATION CONVENTIONAL BIOCHEMICALS HIGH PRESSURE LIQUID CHROMATOGRAPHY ( HPLC) DNA PROBES CULTURE ID DIRECT SPECIMEN TESTING NUCLEIC ACID AMPLIFICATION GUIDELINES FIRST PUBLISHED IN 1996 UPDATED 2000 UPDATED 2009 NAA TESTING SHOULD BE PERFORMED ON AT LEAST ONE RESPIRATORY SPECIMEN FROM EACH PATIENT WITH SIGNS AND SYMPTOMS OF PULMONARY TB FOR WHOM A DIAGNOSIS OF TB IS BEING CONSIDERED, BUT HAS NOT YET BEEN ESTABLISHED, AND FOR WHOM THE TEST RESULT WOULD ALTER CASE MANAGEMENT OR TB CONTROL ACTIVITIES. MMWR: VOL. 58, NO. 1 PP. 7-10, JAN. 16,

13 NAA TESTING NAA RESULT IS POSITIVE AFB SMEAR POSITIVE PRESUME PATIENT HAS TB AND BEGIN TREATMENT. NAA RESULT IS POSITIVE - AFB SMEAR NEGATIVE CLINICAL JUDGEMENT, COLLECT MORE SPECIMENS NAA RESULT IS NEGATIVE AFB SMEAR POSITIVE INHIBITORS PRESENT THAT PREVENT AMPLICICATION. NAA RESULT IS NEGATIVE - AFB SMEAR NEGATIVE CLINICAL JUDGEMENT WHEN SUSCEPTIBILITY - FIRST ISOLATE FROM EACH PATIENT - CULTURE FAILS TO CONVERT TO NEGTIVE AFTER 3 MONTHS OF THERAPY - KNOWN CASES OF THERAPEUTIC FAILURE 13

14 SUSCEPTIBILITY CONT D PROCEDURES DIRECT TESTING 3 WEEKS INDIRECT TESTING 3 WEEKS BROTH CULTURES ONCE BOTTLES BECOME POSITIVE TESTING CAN BE PERFORMED FROM THE ORGANISMS GROWING IN THE BOTTLES. IN THIS CASE RESULTS WILL BE AVAILABLE IN < 7 DAYS. ANTIBIOTICS TESTED TRADITIONAL -ISONIAZID (INH) 2 CONC. -RIFAMPIN - ETHAMBUTOL -PYRAZINAMIDE ( PZA ) 14

15 ANTIBIOTICS CONT D NEW - RIFABUTIN MTB - RIFOPENTINE MTB -AMINOGLYCOSIDES MTB AND NTM AMIKACIN KANAMYCIN STREPTOMYCIN CAPREOMYCIN VIOMYCIN -CYCLOSERINE ALL -ETHIONAMIDE MTB -DAPSONE M. leprae - QUINOLONES MTB, VARIABLE MAC AND RAPID GROWERS -p-aminosalicyclic ACID ( PAS) - MTB DRUG RESISTANCE LUNG TB GREATEST POPULATION ARE THOSE IN CAVITIES (10 7 TO 10 9 ) CASEOUS FOCI MOST COMMON (10 2 TO 10 4 ) ORGANISMS MDR (INH AND RIF) ACCUMULTIVE MUTATIONS RATHER THAN ACQUISTIONS OF AN MDR TRANSFER 15

16 DRUG RESISTANCE RESISTANCE APPEARING DURING TREATMENT IS DUE TO SELECTION AND MULTIPLICATION OF THE RESISTANT MUTANTS PREEXISTING IN THE POPULATION OF THE BACTERIA. SPECIAL POPULATIONS - INH, RMP, SM RAPIDLY GROWING IN PULMONARY LESIONS -RMP GROWING IN SHORTS METABOLIC SPURTS -PZA RESIDE IN ACIDIC ENVIRONMENT OF CASEOUS LESION -NITROMIDAZOLES DORMANT NONREPLICATING DRUG RESISTANCE MULTIPLE DRUG USE IS EFFECTIVE IN ERRADICATION OF TUBERCLE BACILLI WITHIN EACH SPECIAL POPULATION AIM OF MULTIPLE DRUG USE IS TO PREVENT DRUG RESISTANCE AND ACHIEVE MAXIMUM THERAPEURIC EFFECT 16

17 CDC GENOTYPING PROGRAM TWO CDC FUNDED LABORATORIES PERFORM GENOTYPING FOR ONE ISOLATE FROM EVERY CULTURE POSITIVE TB CASE IN US. LABORATORIES SUBMIT ISOLATES TO GENOTYPING LABS GENOTYPING LABS REPORT INITIAL RESULTS TO TB PROGRAMS WITHIN 10 DAYS CALIFORNIA AND MICHIGAN SCIENCE OF GENOTYPING BASED ON ANALYSIS OF DNA IN ALMOST ALL CASES EACH NEW BACILLUS HAD IDENTICAL DNA HOWEVER, CHANGES CAN OCCUR AT LOW FREQUENCY AND THIS CAN LEAD TO DIVERSITY OF THE STRAIN 17

18 GENOTYPING CONT D HOW USED - IDENTIFY INSTANCES OF RECENT TRANSMISSION OR CHAINS OF TRANSMISSION - CAN HELP DIFFERENTIATE REACTIVATION OR REINFECTION WITH NEW STRAIN - IDENTIFY FALSE POS CULTURE LABORATORY DRIVEN PHYSICIAN DRIVEN INTERFERON GAMMA-RELEASE ASSAYS (IGRAS ) USES LATENT TUBERCULOSIS INFECTION ( LTBI ) CURRENT ASSAYS AVAILABLE QUANTIFERON TB GOLD CELLESTIS QUALNTIFERON TB GOLD IN TUBE - CELLESTIS T-SPOT.TB OXFORD IMMUNOTEC 18

19 PRINCIPLE MEASURES T-CELLS RESPONSES TO MYCOBACTERIUM ANTIGENS ESAT-6 6AND CFP-10 BY MEASURING THE RELEASE OF GAMMA INTERFERON. QUANTIFERON DETECTED BY EIA T-SPOT SPOT.TB TB CAPTURES GAMMA INTERFERON IN VICINITY OF T- CELLS SPECIMEN COLLECTION QUANTIFERON TB GOLD MINIMAL 5 ML BLOOD GRENIER VACUETTE LITHIUM HEPARIN TUBE QUANTIFERON TB GOLD IN TUBE 3 TUBES 1 ML EACH VOLUME MUST BE 1 ML AND MUST SHAKE LIKE CRAZY. T-SPOT.TB SODIUM CITRATE OR SODIUM HEPARIN VACUTAINER CPT TUBE (BD) LITHIUM HEPARIN TUBE AND THEN SEPARATE IN LAB USING FICOLL-PAQUE TECHNIQUE ADULTS AND CHILDREN 10 YO AND OVER ONE 8 ML TUBE OR TWO 4 ML CPT TUBES OR TWO LITHIUM HEPARIN 6 ML TUBES CHILDREN 2-9 ONE 4 ML TUBE (BOTH METHODS) CHILDREN < 2 YO ONE 2 ML PEDIATRIC TUBE (BOTH METHODS). 19

20 COLLECTION TIME TO RECEIPT TIME IN LABORATORY QUANTIFERON TB GOLD 12 HOURS FROM COLLECTION AMBIENT TEMP. SHIPMENT QUANTIFERON TB GOLD IN TUBE COLLECTION MUST BE IN 37 C INCUBATOR WITHIN 16 HOURS OF COLLECTION. THIS CAN BE ACCOMPLISHED IN HOUSE OR SENT TO LABORATORY TO INCUBATE. MUST INCUBATE 16 TO 24 HOURS AND THEN CAN BE TESTED OR STORED AT 2 8 C FOR UP TO 3 DAYS. T-SPOT.TB SHOULD BE PROCESSED WITHIN 8 HOURS OF COLLECTION AND STORED AT AMBIENT TEMPERATURE OXFORD HAS VALIDATED A 32 HOUR WINDOW AND EACH LABORATORY WOULD BE REQUIRED TO PROCEED ACCORDINGLY IN THEY WANTED TO USE THAT WINDOW OF 32 HOURS. RESULT INTERPRETATION QUANTIFERON POSITIVE MYCOBACTERIUM TUBERCULOSIS LIKELY NEGATIVE M. TUBERCULOSIS NOT LIKELY. INDETERMINATE CHECK IMMUNE STATUS OF PATIENT AND TECHNICAL ERRORS 20

21 CON T T-SPOT. TB POSITIVE M. TUBERCULOSIS LIKELY NEGATIVE M. TUBERCULOSIS NOT LIKELY BORDERLINE REPEAT TEST INVALID REPEAT TEST REFERENCES 1. MANUAL OF CLINICAL MICROBIOLOGY, V.1, 8 TH EDITION, 2003, ASM PRESS. 2. CLINCIAL MICROBIOLOGY PROCEDUES HANDBOOK, V. 2, 2 ND EDITION, 2004, ASM PRESS 3. MATHEMA, B., N, KUCEPINA, P. J. BIFANI, B.N. KREISWIRTH MOLECULAR EPIDEMIOLOGY OF TUBERCULOSIS: CURRENT INSIGHTS. CLINCIAL MICROBIOLOGY REVIEWS. 19: PP GUIDE TO THE APPLICATIONS OF GENOTYPING TO TUBERCULOSIS PREVENTION AND CONTROL. PREPARED BY NATIONAL TUBERCULOSIS CONTROLLERS ASSOCIATION/CENTERS FOR DISEASE CONTROL AND PREVENTION, ADVISORY GROUP ON TUBERCULOSIS GENOTYPING. JUNE NAVIN, R.T. CDC TB GENOTYPING PROGRAM OVERVIEW. JUNE PACKAGE INSERT T-SPOT.TB JULY 30, 2008, OXFORD IMMUNOTEC 7. PACKAGE INSERT QUANTIFERON TB GOLD AND QUANTIFERON TB GOLD IN TUBE CELLESTIS, INC. 21

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