el futuro de la biología molecular para el diagnóstico...
TRANSCRIPT
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Suzane Silbert, Ph.D. Clinical Scientist
Esoteric Testing Lab/Pathology Department Tampa General Hospital
Tampa, FL. USA.
El futuro de la Biología Molecular para el Diagnóstico Clínico
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PCR:
� Most common method used at the Molecular Lab
� Faster turnaround time than culture based methods
� A lot of advances were noticed since first described
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Three Different Steps:
1. Manual Extraction
2. Amplification
3. Detection
• Whole Procedure: 1 – 3 days • Quantitative results very difficult/detection not precise
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Two Different Steps:
1. Manual Extraction
2. Amplification/ Detection
• Whole Procedure: 1 – 2 days • Quantitative results: standard curves
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Two Different Steps:
1. Automated Extraction
2. Amplification/ Detection
• Results: same day • Quantitative results: standard curves
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Isn’t that GREAT?
1. Automated Extraction
2. Amplification/ Detection
• Results: same day • Quantitative results: standard curves
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It can always get better!!!
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1. Extraction
2. Amplification
3. Detection
3 Instruments/ Steps
or or or
Molecular Diagnostic:
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Why?
One Instrument, One Step:
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Why One Instrument, One Step?
Faster Results Anticipate Treatment
Reduce Length of Hospital Stay
Reduce Overall Costs
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Why One Instrument, One Step?
Faster Results Anticipate Treatment
Reduce Length of Hospital Stay
Reduce Overall Costs
Save Lives
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Why One Instrument, One Step?
Save Lives
Especially your Boss Life… Always under Saving Costs Pressure!!!
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“Sample in – Results out”
Molecular Biology: 1 instrument, 1 step
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GeneXpert:
GX-1
GX-16
GeneXpert Infinity-48
GeneXpert® Module GX-2 GX-4
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Cartridge:
All Testing Done Within Cartridge
� Sample Prep
� Amplification
� Detection
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Vortex and Dispense Sample into Port S
2
Insert Cartridge and Start Assay
3
Insert Swab into Elution Reagent Vial and
Break at Score
1
Total Hands-On time <1 Minute. Results in ~ 1h.
GeneXpert: Procedure
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Women’s Health
GeneXpert Test Portfolio: By the end of 2014
Healthcare Associated Infections
Critical Infectious Disease
CLIA-Waived Testing
Oncology & Genetics
HemosIL FII & FV
Virology
Between Now & 2014 • Targeting 10 New Tests Commercially Available,
Bringing Total to 25 Tests
MRSA/SA BC
SA Nasal Complete
vanA for VRE
MRSA/SA SSTI
MRSA C. difficile
C. diff/Epi
EV Flu GBS CT/NG SA Flu A/B Panel
HIV Quant
BCR/ABL Monitor
V2
HPV BCR/ABL Monitor
V1 CT/NG
Norovirus
MRSA/SA SSTI
MTB/RIF Breast CA Rx Strat
HCV Quant
HBV Quant
Vaginitis
CE-IVD
CE-IVD CE-IVD CE-IVD CE-IVD
MDRO
CE-IVD
Bladder Cancer Monitor
CE-IVD
CT
Vaginitis Vaginosis
CE-IVD
CE-IVD
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EV Flu
MTB/RIF
GeneXpert Test Portfolio: By the end of 2016
Healthcare Associated Infections
Critical Infectious Disease
CLIA-Waived Testing
Oncology & Genetics
Virology
Microbial ID
Candidemia
HIV Quant HCV Quant MRSA/
SA BC
SA Nasal Complete
vanA for VRE
MRSA/SA SSTI
MRSA C. difficile
C. diff/Epi Norovirus
Gastro B Panel
BCR/ABL Monitor
V2 HemosIL FII & FV
BCR/ABL Monitor V1
Breast CA RX Strat
Breast Cancer Metastasis
Risk
SA
CT/NG
Flu A/B Panel
MRSA/SA SSTI EBV Quant
BKV
HBV Quant
HCV Genotype
Group A Strep
Vaginitis Group A Strep
MDRO VAP MRSA/SA
VAP Gram Negative
RSV
CMV Quant
VZV
CE-IVD
CE-IVD CE-IVD
CE-IVD CE-IVD CE-IVD
Vaginitis/Vaginosis
CE-IVD
CE-IVD
46 Tests Available Globally
Bladder Cancer Monitor
CE-IVD
Bladder Sympt
GBS CT/NG
HPV
CE-IVD
CT
Vaginitis Vaginosis
CE-IVD
HSV Typing Lesion
Women’s Health
CE-IVD
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FilmArray – BioFire (Idaho Technologies)
Sample in Results out
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Hands-on-Time: 2 Minutes
Add Sample to Pouch
Add Buffer to Pouch
FilmArray: Procedure
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The FilmArray Pouch/Cartridge:
Reagent Storage
Chemical Circuit Board
Sample Extraction & Purification
1st Stage Multiplex
PCR
2nd Stage PCR
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Results = 1h
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FilmArray:
1. Adenovirus
2. Coronavirus HKU1
3. Coronavirus NL63
4. Human Metapneumovirus
5. Influenza A
6. Influenza A H1
7. Influenza A H1 2009
8. Influenza A H3
9. Influenza B
10. Parainfluenza 1
11. Parainfluenza 2
12. Parainfluenza 3
13. Parainfluenza 4
14. Respiratory Syncytial Virus
15. Rhinovirus /Enterovirus
Respiratory Virus Panel:
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FilmArray:
1. S. aureus
2. S. coagulase neg
3. L. monocytogenes
4. Enterococcus sp
5. Streptococcus sp
6. S. pneumoniae
7. S. agalactiae
8. S. pyogenes
Positive Blood Cultures: 9. Enterobacteriaceae
10. E. cloacae
11. E. coli
12. K. pneumoniae
13. S. marcescens
14. Acinetobacter sp
15. A. baumanii
16. H. influenzae
17. H. flu non typeable
18. N. meningitidis
19. P. aeruginosa
20. C. albicans
21. C. glabrata
22. C. krusei
23. C. parapsolosis
24. C. Tropicalis
25. mecA
26. vanA
27. vanB
28. KPC
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Focus: “Direct Amplification Disc” - DAD
Sample in Results out 8 samples/disc
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Load Sample
Load Reaction Mix
Place disc on instrument; Results available in ≤ 60
minutes
Swab in Transport Media
Transfer Pipette
Required Materials
Reaction Mix
Focus: Procedure
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PCR Setup
0.5 hours
PCR Run
1.5 hours
Total Time
4 hours
Extraction Room PCR Setup Amplification Room
“Traditional” Assay
Assay Setup 2 min
PCR
30-60 min Total Time
≈1 hour
Performed in Single Room/Area
“Sample to Answer” Assay
Sample Prep DNA Extraction
2 hours
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Tests Format Status
Flu A/B & RSV Direct Amplification Disc FDA cleared, 2012
HSV-1 & 2 Direct Amplification Disc FDA Submission, 2013
MRSA Direct Amplification Disc FDA Submission, 2013
Group A Streptococcus
Direct Amplification Disc
FDA Submission 2013
Group B Streptococcus
Direct Amplification Disc In Development
M. tuberculosis Direct Amplification Disc In Development
HIV Direct Amplification Disc In Development
Tests:
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All of these 3 instruments are GREAT:
� Excellent Sensitivity and Specificity � Fast TAT � Reliable � Not Labor Intensive + Walkway
Number of tests available
Cost
Open System
GeneXpert 10 > 20 Expensive No
Filmarray 2 Panels Expensive (?) No
Focus < 5 Less Expensive No
LIMITATION
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BD MAX™:
Only Open System: Sample in Results out
BD MAX™:
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Reagents:
Open System
Extraction
Master Mix IVD
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PCR: Microfluidic Cartridge
• 24 lanes per cartridge • Each lane capable of performing an independent PCR reaction • Supports multiple PCR technologies:
• TaqMan® • Scorpions®/Molecular Beacon • SYBR® Green • Melt Curve analysis • Quantitative Molecular Analysis
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Overview of BD MAX Operation:
Pipet the Sample
Load reagents and specimens
Place racks and cartridges on
BD MAXTM
Create worklist
Close the door to initiate run
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Tampa General Hospital x BD MAX:
October, 2011
May, 2012
March, 2013
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Tampa General Hospital x BD MAX:
1° Validation: Streptococcus β-hemolitico do grupo B – GBS
• Total= 75 clinical samples tested
• Vaginal and rectal swabs collected and sent to the lab for GBS PCR testing
• In the lab, swabs were inoculated in LIM broth and incubated for >18h at 37°C
• Two tests were performed and the results were compared:
Cepheid SmartGBS® x BD MAXTM GBS
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Tampa General Hospital x BD MAX:
1° Validation: Streptococcus β-hemolitico do grupo B – GBS
Positive Negative
Positive 48
0
Negative 3 24
BD MAX™ GBS Sm
artG
BS®
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Tampa General Hospital x BD MAX:
1° Validation: Streptococcus β-hemolitico do grupo B – GBS
• S. pyogenes • E. faecalis • S. pneumoniae • S. aureus • S. epidermidis • E. coli • K. pneumoniae • E. cloacae • S. marcescens • P. aeruginosa • C. albicans • C. tropicalis • N. gonorrheae • A. baumannii • HSV-1 e HSV-2 Total = 16 diferent species tested. All of them negative for GBS
Specificity Test
Dilution BD MAXTM SmartGBS®
1/100,000 Positive Positive
1/1,000,000 Positive Positive
1/10,000,000 Positive Negative
1/100,000,000 Negative Negative
1/1,000,000,000 Negative Negative
Limit of Detection
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Tampa General Hospital x BD MAX:
1° Validation: Streptococcus β-hemolitico do grupo B – GBS
• The BD MAX™ GBS assay has excellent sensitivity and specificity
• The BD MAX™ GBS assay also demonstrated greater analytic
sensitivity based on the LOD and appeared to be more sensitive in detecting GBS positive clinical samples than the Cepheid SmartGBS® assay
• Finally, the BD MAX™ GBS assay offer a “sample in - results
out” format of test, allowing faster results and a less labor intensive procedure
Conclusions:
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Tampa General Hospital x BD MAX:
2° Validation: Staphylococcus aureus resistant to Methicilina - MRSA
• Total= 247 clinical samples tested
• Nare swabs collected and sent to the lab for MRSA PCR testing
• 2 swabs/patient were collected – 1 for each test: GeneXpert MRSA® x BD MAXTM MRSA
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Tampa General Hospital x BD MAX:
2° Validation: Staphylococcus aureus resistant to Methicilina - MRSA
Positive Negative
Positive 30 1
Negative 12(1) 204
BD MAX™ MRSA
Gen
eXpe
rt M
RSA®
1) 5 samples tested by an “in-house” PCR had positive results.
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Tampa General Hospital x BD MAX:
2° Validation: Staphylococcus aureus resistant to Methicilina - MRSA
• CoNS • S. pneumoniae • A. viridans • M. luteus • Group A Streptococcus • Group B Streptococcus • H. influenzae • C. albicans • E. faecalis • Influenzae virus • RSV • Human metapneumovirus • Rhinovirus • Coronavirus • Enterovirus
Total = 15 different species. All of them negative for MRSA
Specificity Test
Dilution BD MAXTM GeneXpert®
1/1,000 Positive Positive
1/10,000 Positive Positive
1/100,000 Positive Positive
1/1,000,000 Negative Negative
1/10,000,000 Negative Negative
Limit of Detection
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Tampa General Hospital x BD MAX:
2° Validation: Staphylococcus aureus resistant to Methicilina - MRSA
• The BD MAX ™ MRSA assay was demonstrated to have an excellent sensitivity, specificity and LOD for MRSA isolates
• Out of 247 samples tested, 13 had different results when both assays were compared.
• One sample was positive by the GeneXpert and negative by the the BD MAXTM and 12 samples were negative by the GeneXpert and positive by the BD MAXTM.
• Five out of the 12 samples positive just by the BD MAXTM were also tested by an in-house PCR test. All of them were positive for MRSA.
• The BD MAX™ MRSA assay appeared to be at least as sensitive in detecting MRSA positive samples than the GeneXpert MRSA® assay.
Conclusions:
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Tampa General Hospital x BD MAX:
3° Validation: Clostridium difficile
• Total= 137 clinical samples testes
• 1 loose/liquid stool container was received for C. difficile PCR testing
• Sample were divided for 2 tests: GeneXpert C. difficile® x BD MAXTM C. difficile
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Tampa General Hospital x BD MAX:
3° Validation: Clostridium difficile toxin B
Positive Negative
Positive 37 4
Negative 1 95
BD MAX™ C. difficile
Gen
eXpe
rt C
. diff
icile
®
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Tampa General Hospital x BD MAX:
3° Validation: Clostridium difficile toxin B
E. coli, E. faecium, E. cloacae
E. faecalis, S. marcescens,
K. pneumoniae, C. albicans,
P. aeruginosa, B. fragilis,
S. aureus, S. epidermidis,
S. sonnei, S. enteritica,
V. parahemolyticus
C. jejuni, Adenovirus 41
Coxsackie A15, Echovirus 27
L. monocytogenes, C. perfringens,
C. sordelii, C. sporogenes
Total = 22 different species. All of them negative for C.difficile
Specificity Test
Dilution BD MAXTM GeneXpert®
1/10 Positive Positive
1/100 Positive Positive
1/1,000 Positive Positive
1/10,000 Positive Positive
1/100,000 Negative Negative
1/1,000,000 Negative Negative
Limit of Detection
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Tampa General Hospital x BD MAX:
3° Validation: Clostridium difficile toxin B
• Both, Cepheid GeneXpert toxin B PCR and Clostridium difficile toxin B had similar performance in detecting C. difficile toxin B genes in stool samples.
• There were 4 samples positive with the GeneXpert and negative with the BD MAXTM.
• These 4 samples had been frozen and thawed and all had cycle numbers on the GeneXpert at 35 or higher.
• The sample that was positive on the BD MAXTM was a fresh sample and was positive at 37 cycles.
• The BD MAXTM represents an effective option for detection of C. difficile with good sensitivity and specificity. The system allows for simple sample in – answer out processing of up to 24 samples in about 2 hours.
Conclusions:
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Collaboration Study:
BD MAX™
Develop and Validate a Test to Detect the Presence of VRE in Rectal Swab Samples
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Collaboration Study:
BD MAX
Develop and Validate a Test to Detect the Presence of VRE in Rectal Swab Samples
• Sent primer sequences to BioGX
• Have BD MAX™ and clinical samples
for future validation
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Collaboration Study:
• Worked on TGH primers and on dual
GeneTAG probe sequences targeting different
regions within the flanking primers
• Custom manufacturing of the test using the
BioGX technology
Develop and Validate a Test to Detect the Presence of VRE in Rectal Swab Samples
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New LDT vanA Assay:
• TGH LDT primers and probes were custom mixed
and lyophilized with buffer, dNTPs, MgCl2 and
enzyme, in a pellet sufficient for 8 reactions
• This BioGX Sample-Ready™ (SR) reagents include
all necessary components for amplification on the
BD Max platform
• The lyophilized pellet is rehydrated with
molecular grade water
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New LDT vanA Assay:
• A Sample Prep Control (SPC)/internal
amplification control (IAC) reagent is also included.
• LDT primers and probes are included in the
lyophilized PCR reagents for detection of this SPC
DNA sequence as well
• The SPC cake has to be rehydrated in 900µL of
molecular grade water.
• It includes a blue dye for visual verification of its
addition
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Extraction Tube"PCR Tube"
1"2"3"
Tube position"
Procedure on the BD MAX:
12.5µL of mastermix was dispensed into microtubes that
fit into the BD MAX strip
3"
Extraction Tube
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Results Analysis:
Color of Detection = 5; Reading Channels= 6 (1 for HRM)
Color Dye Wavelength – Reading Channels
Green FAM 475/520
Yellow TET, VIC 530/565
Orange Texas Red, ROX 585/630
Red Cy5 630/665
Crimson Cy5.5 680/715
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Results – PCR Analysis:
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Results – LDT Test:
Examples of vanA target results, displayed in the 530/565 channel (vanA P1)
Examples of vanA target results, displayed in the 585/630 channel (vanA P2)
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Results – LDT Test:
Examples of SPC/IAC results, displayed in the 680/715 channel (SPC)
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Extraction Tube"PCR Tube"
1"2"3"
Tube position"
Validation:
• 182 clinical rectal swab samples were tested
• Specimens were collected either by ESwabs (Copan) or Traditional
Swabs (LQ Stuart, BD BBL).
• In the lab, both types of swabs were vortexed in 1mL of liquid Amies
(ESwab medium)
• After vortexing, 250µL of the liquid Amies was added to the Expert
vanA buffer and 500µL of the solution was added to the BD EXK3
extraction buffer tube, to provide approximately the same volume of
sample processed in extraction on both systems
• The GeneXpert vanA assay was processed using the procedure
specified in the package insert
• LDT vanA on the BD MAXTM was processed as described
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Extraction Tube"PCR Tube"
1"2"3"
Tube position"
Specificity test: • S. pyogenes
• vanA negative E. faecalis
• vanA negative E. faecium
• P. anaerobius
• S. aureus
• S. epidermidis
• E. coli
• K. pneumoniae
• E. cloacae
• S. marscescens
• P. aeruginosa
None of the strains tested for specificity was positive using the BD MAX™
vanA LDT
• P. mirabilis
• C. albicans
• B. fragilis
• A. baumanii
• L. monocytogenes
• C. difficile
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Results:
GeneXpert LDT Test:
Positive Negative
21 161
Positive Negative
24 158
• All included samples positive on the GX reported positive on the BD MAX
• Three samples reported negative on the GX reported positive on the BD
MAX using the LDT
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Extraction Tube"PCR Tube"
1"2"3"
Costs:
• TGH: 1,400 VRE PCR tests a month
• New LDT vanA test on the BD MAX costs $11.39
less than the vanA PCR test used before
• This represents a saving of:
$15,946.00 /month or $191,352.00 /year
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Extraction Tube"PCR Tube"
1"2"3"
Tube position"
Conclusions:
• The new LDT for vanA on the BD MAX™ has excellent sensitivity and specificity
• It is a user friendly test: “sample in - results out”!
• Requires minimal hands-on time
• Provides fast results
• Represents a significant cost savings
• It is an excellent alternative method for VRE detection in clinical samples
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Extraction Tube"PCR Tube"
1"2"3"
Tube position"
Examples LDT in Development:
• Trichomonas vaginalis
• Enterovirus + Parechovirus
• Cystic Fibrosis Panel
• HBV
• Atypical pneumonia • GI Tract
• And more…
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Tampa General Hospital
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Tampa General Hospital
TGH: • 1.018 beds • 6.900 employees
• Afiliated to University of South Florida – USF • 2012: Named by “America’s Best Hospitals” among the best hospitals in 9 specialties
• Among the leaders of solid transplants in USA
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TGH – Esoteric Testing Lab
1. CMV – quantitative 2. HSV- detection and typing 3. HIV Viral Load 4. HIV qualitative 5. HCV Viral Load 6. Chlamydia trachomatis 7. Neisseria gonorrheae 8. Mutations Factor V Leiden, Factor II e MTHFR 9. Molecular Typing of Microrganismos 10. MRSA 11. Mycobacterium species, 12. M. avium/intracellulare 13. M. tuberculosis 14. Epstein-Barr Virus Quantitative 15. B e T-cell rearrangements 16. BK e JC polyomavirus 17. Bordetella pertussis 18. Parvovirus B19 19. Enterovirus 20. Influenza A/B 21. Influenza A Typing 22. RSV
23. Human metapneumovirus 24. Parainfluenza virus 25. Adenovirus 26. Rhinovirus 27. Group B Streptococcus 28. Mycoplasma pneumoniae 29. JAK2 mutation 30. KPC 31. vanA 32. C. difficile
33. Respiratory Painel: FluA/B, RSV, Paraflu 1-4, Coronavirus HKU1 and NL63, Adenovirus, Rhinovirus, Enterovirus, HMPV,
34. Multiplex for carbapenemases 35. Multiplex for metalo-betalactamases 36. Multiplex for ESBL 37. PCR for qnrs 38. PCR for ampC 39. Varicela Zoster 40. Trichomonas vaginalis
Total mês: ~13.000 exams
Exams Offered/Molecular Diagnostic:
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R&D 1. Suzane Silbert, PhD 2. Carly Kubasek, MS 3. Dom Uy, BA 4. Faris Galambo , BA 5. Nathan Karakad, BA
Ray Widen, PhD – Director
TGH – Esoteric Testing Lab
1. Vicki Healer, MT 2. Alicia Gostnell, MT 3. Elaine Vendrone, MT 4. Laura Ferguson, MT 5. Lisa Wood, MT 6. Lissa Fenton, MT 7. Leah Mashburn, MT 8. Jorge Alves, MT 9. Josh Kay, MT