Infectious Disease April 23, 2015 Bennett Penn, MD/PhD - - PDF document

SLIDE 1

4/23/15 1

New Diagnostic Testing in Infectious Disease

April 23, 2015

Bennett Penn, MD/PhD bennett.penn@ucsf.edu

Image courtesy Wikimedia commons

Division of Infectious Diseases

Disclosures

NONE WHATSOEVER

SLIDE 2

4/23/15 2

Goals: 1) Understand the basic technology 2) Understand advantages/shortcomings of the molecular diagnostics ready for use in primary care setting 3) Understand some of the new technologies becoming available in specialized labs (not ready for widespread use) NOT 1) Comprehensive description of every technology or product on market 2) Endorsement of any particular approach or product

SLIDE 3

4/23/15 3

NOT 1) MALDI-TOF Mass Spectrometry for blood- culture ID’s 2) PCR for blood-culture ID’s 3) Rapid sensitivity testing for blood cultures 4) PCR-MRI for candidemia Case 1

(common but utterly fictional)

You would: A) Admit, start Ceftriaxone, Vancomycin, +/- steroids B) Admit, start Acyclovir, request HSV PCR (2-day turnaround) C) A+B D) Send home 27 y/o female, unremarkable PMH presenting to your ER in Oct with 2d fever 101, HA, photophobia. VS WNL; Exam pt in mild discomfort from HA, somewhat stiff neck, otherwise normal. WBC 10, otherwise nl CBC, Chem. LP: 159 WBC (60% PMN, 40% Lymph) Protein 618, Glucose 55

SLIDE 4

4/23/15 4

Case 2

(quite real)

You would: A) Treat him for TB (INH, RIF, EMB, PZA) B) Treat him for MAC (Azithro, EMB, RIF) C) Treat him for every AFB you can think of (A+B+Aminoglycoside +Imipenem) D) Recommend a second surgery hoping to get micro sample 55 y/o Chinese man who developed ALL. With first chemo, severe pan-

• colitis. Resolves everywhere except at ileocecal junction where persists

despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas -> +AFB on stain. BMT planned for 1-2 mos

Molecular Diagnostics in ID Offer Multiple Advantages

1) Identify unculturable organisms (viruses, certain bacteria) 2) Identify organisms not isolated (often prior antibiotics) 3) Rapidly identify organisms that grow slowly (TB) 4) Point-of-care testing (?)

SLIDE 5

4/23/15 5

A Brief Word About the Technologies

Ø Most techniques rely on detection of DNA/RNA from pathogen Ø Workhorse for this is Polymerase Chain Reaction (PCR)

Heat DNA, Annealing of designed primers Thermostable polymerase

Repeat 30-40 times

Things to Notice About PCR:

Only need to know tiny part of sequence Massive amplification: (~Trillion-fold) Specific Sequence In middle

SLIDE 6

4/23/15 6

Multiplexing

* *

Fluorescent chemical probes can be used to detect different PCR products Can do 5-10 sensors in same tube

Image courtesy pubzi.com

PCR Evolution

Images courtesy Wikimedia commons; clker.com; pixgood.com

SLIDE 7

4/23/15 7

FDA Approved Tests 160+ PCR-based Tests

http://www.fda.gov/Medical Devices/ProductsandMedicalProcedures/InVitroDiagnostics/ ucm330711.htm#microbial

Full list at:

Ø Gonorrhea, Chlamydia (since 1996!) Ø Influenza, RSV, Adenovirus, numerous other respiratory viruses Ø C. Dif Ø HSV-1,2 Ø Enterovirus Ø TB Ø Panels of respiratory pathogens (viruses + bacteria) Ø Panels of bacteria in blood cultures Ø Panels of bacteria in GI infections

Does All This Stuff Actually Work?

Boehme et al NEJM 2010

SLIDE 8

4/23/15 8

Does All This Stuff Actually Work for Detecting Respiratory Viruses?

• No. of

specimens DFA/culture result RVP test result

123 Positive Positive 105 Negative Negative 5 Positive Negative 14 Negative Positive

PCR 123/128+ (97% Sens) Culture 100% Culture PCR

IF “gold-standard” is viral culture

Mahony et al JCM 2007

Does All This Stuff Actually Work?

tested, the RVP assay detected between 0.1 and 100 TCID50 of virus. The RVP test had the following analytical sensitivities: 0.1 TCID50 for rhinovirus, enterovirus, CoV 229E, and influ- enza A virus subtypes H1 and H3; 0.5 TCID50 for influenza B virus, parainfluenza virus type 3, and MPV; 1 TCID50 for RSV type A and parainfluenza virus type 4; 10 TCID50 for parainfluenza virus type 2, RSV type B, and CoVs NL63 and OC43; and 100 TCID50 for adenovirus, parainfluenza virus type 1, and SARS-CoV. The corresponding analytical sen- sitivities in genome equivalents were 50 to 250 for all virus types/subtypes. We evaluated the performance of the RVP assay by testing 294 respiratory tract specimens that were submitted to the clinical virology laboratory for routine investigation

• f

respi- ratory viruses. Aliquots of each specimen were tested by rou- tine DFA plus culture, followed by the RVP test. DFA and culture were performed in the clinical virology laboratory, and the RVP test was performed in the research laboratory by technologists blinded to previous results obtained for the spec- imens. For the 294 specimens, there were 228 concordant results, including 123 positives by DFA/culture and the RVP test and 105 negatives by both tests (Table 2). DFA/culture detected 128 positive specimens, and the RVP test detected 123

• f

these, for an unadjusted sensitivity

• f

96.1% for the seven conventional respiratory viruses (influenza A and B vi- ruses, parainfluenza virus types 1 to 3, RSV, and adenovirus) routinely detected in most clinical laboratories. The RVP test detected an additional 61 positive specimens, 14 of which were negative by DFA/culture for the seven viruses tested, and 47 were positive for viruses not tested for by DFA/culture. These 61 additional positive specimens included 2 for influenza A virus, 1 for parainfluenza virus type 1, 2 for parainfluenza virus type 2, 1 for parainfluenza virus type 4, 2 for RSV, 8 for MPV, 39 for rhinovirus/enterovirus, 6 for OC43 CoV, 2 for NL63 CoV, 1 for HKU1 CoV, and 3 specimens that were positive for two viruses, including 1 specimen that was positive for MPV and rhinovirus/enterovirus and 2 specimens that were positive for OC43 and rhinovirus/enterovirus. All of the 66 specimens that gave discordant results, including the 5 DFA/culture-pos- itive specimens that were negative by the RVP test and the 61 specimens that were positive by the RVP test and negative or positive for viruses not tested for by DFA/culture, were tested by a second PCR that targeted a dif ferent area

• f

the viral

• genome. Table 3 shows the results for the 5 specimens that had

given DFA/culture-positive, RVP test-negative discordant re- sults and for the 14 specimens that had given DFA/culture- negative, RVP test-positive discordant results. Three

• f

the 5 DFA/culture-positive, RVP test-negative specimens (numbers 167, 191, and 187) were confirmed to be positive by PCR, indicating two false positives (numbers 286 and 62) by DFA/ culture. All

• f

the additional 61 RVP test-positive specimens were confirmed as true positives by the second PCR. If a true positive is defined as being positive by two or more tests (DFA, culture, the RVP test, and/or confirmatory PCR), then there were 183 positives and 111 negatives. To determine how the RVP test performed compared to DFA and culture, we elim- inated the 47 specimens that were positive for a virus not tested for by DFA and culture (i.e., parainfluenza 4; MPV; CoVs OC43, 229E, NL63, and HKU1; and rhinovirus/enterovirus) and used the remaining 247 specimens for analysis. Among these 247 specimens, there were 137 positives and 110 nega-

• tives. The sensitivity and specificity of DFA/culture were 91.9%

(126/137) and 98.2% (108/110), respectively. The RVP test had a sensitivity

• f

97.8% (134/137) and a specificity

• f

96.4% (107/110). If, however, all confirmed respiratory viruses de- tected by the RVP test are included in the analysis, then the RVP assay detected 180 out of 183 positive specimens and had an overall sensitivity of 98.4%, whereas DFA/culture detected

• nly 126 out of 183 specimens and had a sensitivity of 68.8%.

Of particular interest was the finding that 15

• ut
• f

294 (5.2%) specimens were positive for two viruses in this group of specimens. The dual infections included the following combi- nations:

• ne

type 1 parainfluenza virus plus

• ne

rhinovirus/ enterovirus, one type 2 parainfluenza virus plus one rhinovirus/ enterovirus, two type 3 parainfluenza viruses plus

• ne

rhinovirus/enterovirus, three RSVs plus one rhinovirus/entero- virus,

• ne

adenovirus plus

• ne

rhinovirus/enterovirus,

• ne

MPV plus

• ne

OC43 CoV, three MPVs plus

• ne

rhinovirus/ enterovirus, two OC43 CoVs plus

• ne

rhinovirus/enterovirus, and

• ne

adenovirus plus

• ne

KHU1 CoV. No specimen was positive for three respiratory viruses. Testing additional spec- TABLE 2. Distribution

• f

DFA/culture results and RVP test results for 294 NP specimens

• No. of

specimens DFA/culture result RVP test result 123 Positive Positive 105 Negative Negative 5 Positive Negative 14 Negative Positive 47 Not tested Positive TABLE 3. PCR results for 5 DFA/culture-positive, RVP test-negative and 14 DFA/culture-negative, RVP test-positive specimens Specimen no. Result by testa: DFA/culture RVP (MFI reading) Second PCRb 167 Flu B Flu B (42) Flu B 191 Para 2 Para 2 (13) Para 2 286 Para 2 Para 2 (29.5) Para 2 62 Para 1 Para 1 (20) Para 1 187 RSV RSV (93) RSV 108 Flu A Flu A (298) Flu A 53 Para 2 Para 2 (266) Para 2 349 Para 2 Para 2 (6447) Para 2 89 Para 1 Para 1 (215) Para 1 443 RSV RSV (213) RSV 58 Flu A Flu A (412) Flu A 128 Mpn Mpn (7229) Mpn 441 Mpn Mpn (1837) Mpn 503 Mpn Mpn (286) Mpn 549 Mpn Mpn (656) Mpn 566 Mpn Mpn (966) Mpn 601 Mpn Mpn (6032) Mpn 604 Mpn Mpn (3537) Mpn 119 Mpn Mpn (6772) Mpn a Flu A, influenza A virus; Flu B, influenza B virus; Para 1, parainfluenza virus type 1; Para 2, parainfluenza virus type 2; Mpn, metapneumovirus. b A second confirmatory PCR targeting a unique genomic region was performed to resolve the discordant results, as described in Materials and Methods. 2968 MAHONY ET AL.

• J. CLIN. MICROBIOL.
• n April 18, 2015 by UCSF Library & CKM

http://jcm.asm.org/ Downloaded from

test results for 294 NP specimens

• No. of

specimens DFA/culture result RVP test result

123 Positive Positive 105 Negative Negative 5 Positive Negative 14 Negative Positive 47 Not tested Positive

PCR 123/128+ (97% Sens) Sen 69% Sens 98% Culture PCR

IF “gold-standard” is [culture + PCR] w/ discrep resolved by 3rd test:

SLIDE 9

4/23/15 9

Problems with 1st Generation Molecular Tests:

SLOW and COMPLICATED

Problems: SLOW and COMPLICATED

Extract DNA/RNA from sample (1h) RT Set-up (1h) Run RT (1h) PCR Setup (1h) PCR Run (2-3h)

Image courtesy Wikimedia commons

Basically an entire day

SLIDE 10

4/23/15 10

Simplexa (Focus) GeneXpert (Cepheid) Filmarray (Biofire)

Pouch Plastic Film and Purifjcation

Solution: Engineering

Cartridges: Ø Built-in lysis device (sonicator, beads) Ø Pre-made compartments for adding buffers Ø Pre-made compartments with PCR reagents Ø Optical PCR machine to read signal Ø Fluidics to move sample around for you

2nd Generation Tests: Fully Automated

Extract DNA/RNA from sample (1h) RT Set-up (1h) Run RT (1h) PCR Setup (1h) PCR Run (1-2h) Add single buffer, put in cartridge (5 min) Load Machine (5min) PCR Run (1-2h)

2-3h Basically an entire day

SLIDE 11

4/23/15 11

Do Fully Automated Systems Actually Work? Example #1: TB:

Boehme et al NEJM 2010

Background – TB Diagnosis

1) AFB smear ~60% sensitive for single smear ~80% sensitive if 3 smears Detects 5000 bacilli/ mL Rapid diagnosis (4-6h hands on) 2) AFB Culture ~95% sensitive – ‘gold standard’ Detects 10-100 bacilli/ml Delay of weeks for diagnosis

SLIDE 12

4/23/15 12

Single-Run Sensitivity – About the Same as Culture

Boehme et al NEJM 2010

How Does this Affect Patient Care?

IF PCR is: A) More sensitive AND B) Theoretically faster THEN can it get patients out of airborne isolation/hospital faster?

SLIDE 13

4/23/15 13

2 Small Studies Say It Does

207 “TB Rule-Outs”1 6 TB Patients w/ TB 6/6 Sm+ Cx+ 6/6 PCR+ Cx+

21h 68h PCR Sput

142 “TB Rule-Outs”2 9 TB Patients w/ TB 8/9 Sm+ Cx+ 8/9 PCR+ Cx+

65h Sput

x

34h PCR

1) Chaisson et al Clin Inf Dis 2014; 2) Lippencott Clin Inf Dis 2014

“…The Food and Drug Administration (FDA) has cleared the Xpert MTB/RIF Assay (Cepheid; Sunnyvale, California) with an expanded intended use that includes testing of either one or two sputum specimens as an alternative to examination of serial acid-fast stained sputum smears to aid in the decision of whether continued airborne infection isolation (AII) is warranted…”

Morbidity and Mortality Weekly Report (MMWR)

Revised Device Labeling for the Cepheid Xpert MTB/RIF Assay for Detecting Mycobacterium tuberculosis

Weekly

February 27, 2015 / 64(07);193-193

SLIDE 14

4/23/15 14

You would: A) Admit, start Ceftriaxone, Vancomycin, +/- steroids B) Admit, start Acyclovir, request HSV PCR (2-day turnaround) C) A+B D) Send home 27 y/o female, unremarkable PMH presenting to your ER in Oct with 2d fever 101, HA, photophobia. VS WNL; Exam pt in mild discomfort from HA, somewhat stiff neck, otherwise normal. WBC 10, otherwise nl CBC, Chem. LP: 159 WBC (60% PMN, 40% Lymph) Protein 618, Glucose 55

Case 1 Do Fully Automated Systems Actually Work?

Example #2: Aseptic Meningitis

2nd Generation Enterovirus PCR Test: 434 Patients evaluated for meningitis Ø 6 cases bacterial Ø 107 cases Enteroviral (Gold std: Culture+Other NAAT) PCR: 94% Sensitive 100% Specific

Nolte et al

Nolte et al J Clin Micro 20111

SLIDE 15

4/23/15 15

Do Fully Automated Systems Actually Work?

Example #2: Aseptic Meningitis

Additional Large Cohort (kids): 3200 Patients w/ meningitis Ø 121 w/ Bacterial

• Zero patients (+) for enterovirus

Ø ~3000 ‘aseptic’ 64%+ for enterovirus Again 100% Specificity

Nigrovic et al CID 2010

Fully Automated PCR Diagnosis Can Save Time and Money

No PCR, Neg Bact Manual PCR + Automated PCR+

Empirical

antibiotic administration (%)

Duration

• f

antibacterial therapy, median days (IQR)

Empirical

acyclovir administration (%)

Length

• f

stay, median days (IQR)

Hospitalization

costs, median, $ (IQR)

IQR = interquartile range. Group A(n

= 17)

Group

B(n = 20)

Group

C(n = 22)

11

(64)

14

(70)

12

(55)

1 (0–6) 1 (0–1.9) 0.5

(0–0.5)

8 (47) 4 (20) 4

(2.5–7.5)

2 (1–3.7) 0.5

(0.3–0.7)

5458

(2676–6274)

2796

(2062–5726)

921

(765–1230)

4d/ $5,500 2d/ $2700 12h/ $950

Giulier et al J Clin Virology 2015

SLIDE 16

4/23/15 16

You would: A) Admit, start Ceftriaxone, Vancomycin, +/- steroids B) Admit, start Acyclovir, request HSV PCR (2-day turnaround) C) A+B D) Send home E) Get Enterovirus PCR, send home if + 27 y/o female, unremarkable PMH presenting to your ER in Oct with 2d fever 101, HA, photophobia. VS WNL; Exam pt in mild discomfort from HA, somewhat stiff neck, otherwise normal. WBC 10, otherwise nl CBC, Chem. LP: 159 WBC (60% PMN, 40% Lymph) Protein 618, Glucose 55

Case 1

Table 4. Outcomes Before and After Rapid Respiratory Panel (RRP) Implementation Regardless of Whether the Test Result Was Positive or Negative

Variable Pre-RRP (n ¼ 365) Post-RRP (n ¼ 771) P Value Time to test result, mean (SD), min 1119 (492) 383 (293) ,.001 PCR results received in ED before admission, No. (%) 49 (13.4) 398 (51.6) ,.001 Antibiotic prescribed, No. (%) 268 (73.4) 555 (72.0) .61 Antibiotic use, mean (SD), d 3.2 (1.6) 2.8 (1.6) .003 Inpatient LOS, mean (SD), d 3.4 (1.7) 3.2 (1.6) .16 ED LOS, mean (SD), min 256 (97) 282 (115) .002 Time in isolation, mean (SD), h 73 (41) 70 (38) .27 Abbreviations: ED, emergency department; LOS, length of stay; PCR, polymerase chain reaction.

Example #3: Respiratory Viruses

Pouch Plastic Film and Purifjcation

Rogers et al Arch Path Lab Med 2014

1st Gen 2nd Gen

50% of Results While Patient is in ER

SLIDE 17

4/23/15 17

Pouch Plastic Film and Purifjcation

Rogers et al Arch Path Lab Med 2014

2nd Gen ($60/sample)

D/C Who gets Tamiflu? Who gets Z-Pak? Who gets OJ? Admit Abx/Antivirals? Who needs isolation

Respiratory Panel: 50% of Results While Patient is in ER

Take-Home #1:

Molecular Diagnosis with automated PCR is sensitive, fast, and often cost-effective… … and will likely be coming to hospitals/ER’s/clinics near you soon.

SLIDE 18

4/23/15 18

Part 2: Emerging Technologies

(not quite ready for primary care clinic) 1) “16s sequencing” (“Broad Range PCR”)

2) “Next Generation Sequencing” (“Deep Sequencing”) Technologies to identify difficult-to-identify pathogens

New Techniques for Unidentified Pathogens

1) “16s sequencing” (“Broad Range PCR”)

SLIDE 19

4/23/15 19

16s PCR: Principle

Sequence to get molecular fingerprint of the pathogen

Sequence every single bacteria has Unique sequences each species

16s rRNA: Most highly-conserved gene in nature

Renvoise et al Medecine et maladies infect 2013

16s PCR

ADVANTAGES: Can detect: Hard-to-grow pathogens Slow-growing pathogens rapidly Pathogens from formalin-fixed slides DISADVANTAGES: Complex to perform (few labs) 1-week turnaround Insensitive – not always + even if bacteria present Will not detect viruses

SLIDE 20

4/23/15 20

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.
• Started on RIPE for probable TB

SLIDE 21

4/23/15 21

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.
• Started on RIPE for probable TB
• Portion of formalin-fixed path tissue sent to CA DPH for TB

PCR -> NEGATIVE

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.
• Started on RIPE for probable TB
• Portion of formalin-fixed path tissue sent to CA DPH for TB

PCR -> Negative

• Portion of formalin-fixed path tissue sent to UW for 16s

sequencing

SLIDE 22

4/23/15 22

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.
• Started on RIPE for probable TB
• Portion of formalin-fixed path tissue sent to CA DPH for TB

PCR -> Negative

• Portion of formalin-fixed path tissue sent to UW for 16s

sequencing

• > + M. Avium Complex (MAC)

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.
• Started on RIPE for probable TB
• Portion of formalin-fixed path tissue sent to CA DPH for TB

PCR -> Negative

• Portion of formalin-fixed path tissue sent to UW for 16s

sequencing

• > + M. Avium Complex (MAC)
• > Stopped INH, PZA, started Azithro for MAC

SLIDE 23

4/23/15 23

Case 2 - Continued

55 y/o Chinese man who developed ALL. With first chemo, severe pan-colitis. Resolves everywhere except at ileocecal junction where persists despite several months oral abx (Cipro, Flagyl, Augmentin). Developed a fistula which is resected. Felt by surgeons to be non-infx, no cultures. Path shows granulomas

• > +AFB on stain.

Pt now ~2 months into treatment, feeling much better, BMT pending Several Laboratories offer 16s PCR University of Washington Mayo Clinic Harvard

SLIDE 24

4/23/15 24

Several Laboratories offer 16s PCR University of Washington Mayo Clinic Harvard UCSF Experience thus far:

Ø Samples sent to UW Ø 175 Samples sent (~3 years) Ø 37 Positive Samples (21% positive) 16% 16s PCR 5% Other PCR

Rutishauser-R, Babik-J, and Miller-S (unpublished data)

UCSF Experience 21% positive

Image courtesy iyoodle.com

21% positive vs

SLIDE 25

4/23/15 25

New Techniques for Unidentified Pathogens

1) “16s sequencing” (“Broad Range PCR”) 2) “Next Generation Sequencing” (“Deep Sequencing”)

Next Generation Sequencing

ADVANTAGES:

Can detect: ANY DNA/RNA (including unknown viruses) Theoretically, pathogens from formalin-fixed slides

DISADVANTAGES:

Very, very complex procedure, often several weeks for result Not commercially available; research-only right now, very few labs

SLIDE 26

4/23/15 26

Next Generation Sequencing FANCY STEP 1:

Glue on artificial sequences

GO TO FANCY STEP 2 FANCY STEP 2:

Sequence: Use microscope+computer to track each base added in each cluster

Ross, et al, Am J Clin Pathol 2011;136:527-539

FANCY STEP 3:

Stick to Microscope slide (spread out molecules) PCR-amplify molecules into clusters

GET SEQUENCE OF MILLIONS OF DNA MOLECULES

SLIDE 27

4/23/15 27

Next Generation Sequencing

VERY powerful… but VERY complex Ø Specialized equipment Ø 3+ days of hands-on technician time Ø Complicated bioinformatics manipulations to make sense of 5-10 million DNA sequences

Next Generation Sequencing: Case #3

14 y/o boy, SCID s/p BMT. Few mos HA, now 2 weeks fever, progressive

• AMS. Exposures: cats at home, trip to Puerto Rico 6 mos prior .

Started on Steroids but further AMS->Status epilepticus->Intubated Brain biopsy: granulomas, additional cultures/PCR still negative Started on Cefuroxime, several days without improvement Several LP’s: 120 WBC (L 60%), Protein 120, Glucose 10

• Multiple bacterial, fungal, AFB cultures neg
• Multiple (dozens) of PCR tests for bacterial, viral pathogens neg
• 16s PCR negative x 2 (UW)

SLIDE 28

4/23/15 28

Next Generation Sequencing: Case #3

14 y/o boy, SCID s/p BMT. Few mos HA, now 2 weeks fever, progressive

• AMS. Exposures: cats at home, trip to Puerto Rico 6 mos prior .

Started on Steroids but further AMS->Status epilepticus->Intubated Brain biopsy: granulomas, additional cultures/PCR still negative Started on Cefuroxime, several days without improvement

Samples sent (on research basis) for NGS at UCSF

Several LP’s: 120 WBC (L 60%), Protein 120, Glucose 10

• Multiple bacterial, fungal, AFB cultures neg
• Multiple (dozens) of PCR tests for bacterial, viral pathogens neg
• 16s PCR negative x 2 (UW)

Next Generation Sequencing: Case #3

Samples sent (on research basis) for NGS at UCSF Ø Processing was expedited given critically ill patient, 3d turn-around Ø 3,063,784 total sequences (mostly human) Ø 475 sequences from Leptospira Ø Never seen in any other sample lab had processed

brief report

The new engl and jour nal of medicine

Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing

Michael R. et al NEJM 2014 ,

SLIDE 29

4/23/15 29

Next Generation Sequencing: Case #3

475 sequences from Leptospira

• Very rare cause of meningitis
• Usually serologic diagnosis (vs culture at CDC)

Local physicians notified

• Antibiotics changed to hi-dose IV Penicillin
• Over next 7d:
• Seizures stopped,
• CSF profile started to improve
• Discharged to rehab 2 weeks later for PT

Images courtesy standardsingenomics.org

Next Generation Sequencing

VERY powerful… but VERY complex… …but if harnessed in select settings can reveal a pathogen you wouldn’t find any other way

SLIDE 30

4/23/15 30

Take-Home #2:

16s PCR and Next Generation Sequencing will not be coming to primary care clinics any time soon. HOWEVER: They allow molecular identification of pathogens in previously very difficult ‘culture-negative’ cases.

THANKs!

SLIDE 31

4/23/15 31

References

Boehme, Catharina C, Pamela Nabeta, Doris Hillemann, Mark P Nicol, Shubhada Shenai, Fiorella Krapp, Jenny Allen, et al. 2010. “Rapid Molecular Detection of Tuberculosis and Rifampin Resistance..” New England Journal of Medicine 363 (11): 1005–15. doi:10.1056/NEJMoa0907847. Chaisson, L H, M Roemer, D Cantu, B Haller, A J Millman, A Cattamanchi, and J L Davis. 2014. “Impact of GeneXpert MTB/RIF Assay on Triage of Respiratory Isolation Rooms for Inpatients with Presumed Tuberculosis: a Hypothetical Trial.” Clinical Infectious Diseases 59 (10): 1353–60. doi:10.1093/cid/ciu620. Giulieri, Stefano G, Caroline Chapuis-Taillard, Oriol Manuel, Olivier Hugli, Christophe Pinget, Jean-Blaise Wasserfallen, Roland Sahli, Katia Jaton, Oscar Marchetti, and Pascal Meylan. 2015a. “Rapid Detection of Enterovirus in Cerebrospinal Fluid by a Fully-Automated PCR Assay Is Associated with Improved Management of Aseptic Meningitis in Adult Patients..” Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology 62 (January): 58–62. doi:10.1016/j.jcv.2014.11.001. Giulieri, Stefano G, Caroline Chapuis-Taillard, Oriol Manuel, Olivier Hugli, Christophe Pinget, Jean-Blaise Wasserfallen, Roland Sahli, Katia Jaton, Oscar Marchetti, and Pascal Meylan. 2015b. “Rapid Detection of Enterovirus in Cerebrospinal Fluid by a Fully-Automated PCR Assay Is Associated with Improved Management of Aseptic Meningitis in Adult Patients.” Journal of Clinical Virology 62 (January). Elsevier B.V.: 58–62. doi:10.1016/j.jcv.2014.11.001. Lippincott, C K, M B Miller, E B Popowitch, C F Hanrahan, and A Van Rie. 2014. “Xpert MTB/RIF Assay Shortens Airborne Isolation for Hospitalized Patients with Presumptive Tuberculosis in the United States.” Clinical Infectious Diseases 59 (2): 186–92. doi:10.1093/cid/ciu212. Mahony, J, S Chong, F Merante, S Yaghoubian, T Sinha, C Lisle, and R Janeczko. 2007. “Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay.” Journal of Clinical Microbiology 45 (9): 2965–70. doi:10.1128/JCM.02436-06. Nigrovic, Lise E, Richard Malley, Dewesh Agrawal, and Nathan Kuppermann. 2010. “Low Risk of Bacterial Meningitis in Children with a Positive Enteroviral Polymerase Chain Reaction Test Result.” Clinical Infectious Diseases 51 (10): 1221–22. doi:10.1086/656919. Nigrovic, Lise E, Richard Malley, Dewesh Agrawal, Nathan Kuppermann, Pediatric Emergency Medicine Collaborative Research Committee of the American Academy of Pediatrics. 2010. “Low Risk of Bacterial Meningitis in Children with a Positive Enteroviral Polymerase Chain Reaction Test Result..” Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America 51 (10): 1221–22. doi:10.1086/656919. Nolte, F S, B B Rogers, Y W Tang, M S Oberste, C C Robinson, K S Kehl, K A Rand, H A Rotbart, J R Romero, A C Nyquist, and D H Persing. 2011a. “Evaluation of a Rapid and Completely Automated Real-Time Reverse Transcriptase PCR Assay for Diagnosis of Enteroviral Meningitis.” Journal of Clinical Microbiology 49 (2): 528–33. doi:10.1128/JCM.01570-10. Nolte, Frederick S, Beverly B Rogers, Yi-Wei Tang, M Steven Oberste, Christine C Robinson, K Sue Kehl, Kenneth A Rand, Harley A Rotbart, Jose R Romero, Ann-Christine Nyquist, and David H Persing. 2011b. “Evaluation of a Rapid and Completely Automated Real-Time Reverse Transcriptase PCR Assay for Diagnosis of Enteroviral Meningitis..” Journal of Clinical Microbiology 49 (2): 528–33. doi:10.1128/JCM.01570-10. Rogers, Beverly B, Prabhu Shankar, Robert C Jerris, David Kotzbauer, Evan J Anderson, J Renee Watson, Lauren A O'Brien, Francine Uwindatwa, Kelly McNamara, and James E Bost. 2014. “Impact of a Rapid Respiratory Panel Test on Patient Outcomes.” Archives of Pathology & Laboratory Medicine, August, 140825052632001–7. doi:10.5858/arpa.2014-0257-OA. Ross, J S, and M Cronin. 2011a. “Whole Cancer Genome Sequencing by Next-Generation Methods.” American Journal of Clinical Pathology 136 (4): 527–39. doi:10.1309/AJCPR1SVT1VHUGXW. Ross, Jeffrey S, and Maureen Cronin. 2011b. “Whole Cancer Genome Sequencing by Next-Generation Methods..” American Journal of Clinical Pathology 136 (4): 527–39. doi:10.1309/AJCPR1SVT1VHUGXW. Wilson, Michael R, Samia N Naccache, Erik Samayoa, Mark Biagtan, Hiba Bashir, Guixia Yu, Shahriar M Salamat, et al. 2014. “Actionable Diagnosis

• f Neuroleptospirosis by Next-Generation Sequencing.” New England Journal of Medicine 370 (25): 2408–17. doi:10.1056/NEJMoa1401268.!