Division of Infectious Diseases

New Diagnostic Testing in Infectious Disease April 23, 2015 Bennett Penn, MD/PhD [email protected]

Image courtesy Wikimedia commons

Disclosures

NONE WHATSOEVER

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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

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NOT 1) MALDI-TOF Mass Spectrometry for bloodculture 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) 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 You would: A) Admit, start Ceftriaxone, Vancomycin, +/- steroids B) Admit, start Acyclovir, request HSV PCR (2-day turnaround) C) A+B D) Send home

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Case 2

(quite real) 55 y/o Chinese man who developed ALL. With first chemo, severe pancolitis. 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 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

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 (?)

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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

Specific Sequence In middle Massive amplification: (~Trillion-fold)

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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

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FDA Approved Tests 160+ PCR-based Tests 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

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

Does All This Stuff Actually Work?

Boehme et al NEJM 2010

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Does All This Stuff Actually Work for Detecting Respiratory Viruses? MAHONY ET AL.

2968

IF “gold-standard” culture TABLE 2. Distributionisofviral DFA/culture results and RVP test resultsCulture for 294 NP specimens

No. of specimens

123 105 5 14 47

TABLE 3. PCR results for 5 DFA/culture-positive, RVP test-negative and 14 DFA/culture-negative, RVP test-positive specimens

PCR

DFA/culture result

RVP test result

Positive Negative Positive Negative Culture 100% Not tested

PCR

J. CLIN. MICROBIOL.

Positive Negative Negative Positive 123/128+ Positive

Specimen no.

(97%

DFA/culture

RVP (MFI reading)

Second PCRb

Flu B Para 2! Para 2! Para 1! RSV! Flu A" Para 2" Para 2" Para 1" RSV" Flu A" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn"

Flu B (42) Para 2" (13) Para 2" (29.5) Para 1" (20) RSV" (93) Flu A! (298) Para 2! (266) Para 2! (6447) Para 1! (215) RSV! (213) Flu A! (412) Mpn! (7229) Mpn! (1837) Mpn! (286) Mpn! (656) Mpn! (966) Mpn! (6032) Mpn! (3537) Mpn! (6772)

Flu B! Para 2! Para 2" Para 1" RSV! Flu A! Para 2! Para 2! Para 1! RSV" Flu A" Mpn! Mpn! Mpn! Mpn! Mpn! Mpn! Mpn! Mpn!

!

"

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 influenza 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 sensitivities in genome equivalents were 50 to 250 for all virus types/subtypes. a Flu A, influenza A virus; Flu B, influenza B virus; Para 1, parainfluenza virus et al JCM 2007 WeMahony evaluated the performance of the RVP assay by testing type 1; Para 2, parainfluenza virus type 2; Mpn, metapneumovirus. b 294 respiratory tract specimens that were submitted to the A second confirmatory PCR targeting a unique genomic region was performed to resolve the discordant results, as described in Materials and clinical virology laboratory for routine investigation of respiMethods. ratory viruses. Aliquots of each specimen were tested by routine DFA plus culture, followed by the RVP test. DFA and culture were performed in the clinical virology laboratory, and indicating two false positives (numbers 286 and 62) by DFA/ the RVP test was performed in the research laboratory by culture. All of the additional 61 RVP test-positive specimens technologists blinded to previous results obtained for the specwere confirmed as true positives by the second PCR. If a true imens. For the 294 specimens, there were 228 concordant positive is defined as being positive by two or more tests (DFA, results, including 123 positives by DFA/culture and the RVP culture, the RVP test, and/or confirmatory PCR), then there test and 105 negatives by both tests (Table 2). DFA/culture were 183 positives and 111 negatives. To determine how the detected 128 positive specimens, and the RVP test detected RVP test performed compared to DFA and culture, we elim123 of these, for an unadjusted sensitivity of 96.1% for the inated the 47 specimens that were positive for a virus not tested seven conventional respiratory viruses (influenza A and B vifor by DFA and culture (i.e., parainfluenza 4; MPV; CoVs ruses, parainfluenza virus types 1 to 3, RSV, and adenovirus) OC43, 229E, NL63, and HKU1; and rhinovirus/enterovirus) routinely detected in most clinical laboratories. The RVP test and used the remaining 247 specimens for analysis. Among detected an additional 61 positive specimens, 14 of which were these 247 specimens, there were 137 positives and 110 neganegative by DFA/culture for the seven viruses tested, and 47 tives. The sensitivity and specificity of DFA/culture were 91.9% were positive for viruses not tested for by DFA/culture. These (126/137) and 98.2% (108/110), respectively. The RVP test had 61 additional positive specimens included 2 for influenza A PCR 123/128+ (97% Sens) a sensitivity of 97.8% (134/137) and a specificity of 96.4% virus, parainfluenza virus type 1, 2 for parainfluenza virus 29681 forMAHONY ET AL. J. CLIN. MICROBIOL . (107/110). If, however, all confirmed respiratory viruses detype 2, 1 for parainfluenza virus type 4, 2 for RSV, 8 for MPV, tected by test are in the analysis, 39 “gold-standard” forTABLE rhinovirus/enterovirus, 6 for+OC43 CoV, 2 for NL63 TABLE 3. RVP PCR results forincluded 5 DFA/culture-positive, RVPthen the 2. Distribution of DFA/culture results and RVP IF is [culture PCR] w/ discrep resolved by 3rdthe test: RVP assay test-negative detected 180 out 183 positive specimens and had CoV, 1 for HKU1test CoV, andfor 3 specimens that were positive for and 14 of DFA/culture-negative, results 294 NP specimens Culture PCR RVPof test-positive specimens an overall sensitivity 98.4%, whereas DFA/culture detected two viruses, including 1 specimen that was positive for MPV No. of DFA/culture RVP test result only 126 out of 183 specimensResult and by had and rhinovirus/enterovirus and 2 result specimens that were positive specimens testaa: sensitivity of 68.8%. Specimen Of particular interest was the finding that 15 out of b294 for OC43 and rhinovirus/enterovirus. All of the 66 specimens no. DFA/culture RVP (MFI reading) Second PCR 123 Positive Positive (5.2%) specimens were positive for two viruses in this group of that 105 gave discordant results, including the 5 DFA/culture-posNegative Negative ! " Flu B! 167 Flu B Flu B (42) specimens. The dual infections included the following combiitive 5specimens that were Positive negative by the RVP test and the 61 Negative ! Para 2! 191 Para 2" (13) 14 Negative Positive or nations: one Para type 21! parainfluenza specimens that were positive by the RVP test and negative " virus plus one rhinovirus/ Para 2" 286 Para 2 Para 2 (29.5) 47 Not tested Positive enterovirus, one virus plus onePara rhinovirus/ positive for viruses not tested for by DFA/culture, were tested 1" 62 Paratype 1! 2 parainfluenza Para 1" (20) ! 187 RSV RSV" (93) enterovirus, two ! type 3 parainfluenza virusesRSV plus one by a second PCR that of the98% viral Sentargeted 69% a different area Sens Flu A! 108 Flu A" A! plus (298)one rhinovirus/enterorhinovirus/enterovirus, three Flu RSVs genome. Table 3 shows the results for the 5 specimens that had " ! ! Para 2 53 Para 2 Para 2 (266) tested, the RVP assay detected between 0.1 and 100 TCID50reof virus, one adenovirus plus Para one 2!rhinovirus/enterovirus, one given DFA/culture-positive, RVP test-negative discordant Para 2! 349 Para 2" (6447) " ! virus. The had the following sensitivities: MPV plus one OC43 CoV, three MPVs plus one rhinovirus/ sults and forRVP the test 14 specimens that hadanalytical given DFA/culturePara 1! 89 Para 1 Para 1 (215) " ! 0.1 TCID rhinovirus, discordant enterovirus,results. CoV 229E, RSV" 443 RSVOC43 (213) enterovirus, two CoVsRSV plus one rhinovirus/enterovirus, negative, RVP test-positive Threeand of influthe 5 50 for Flu A" was 58 one adenovirus Flu A" plus oneFlu A! (412) enza A virus subtypesRVP H1 and H3; 0.5 TCID B and KHU1 CoV. No specimen DFA/culture-positive, test-negative specimens (numbers 50 for influenza " ! ! Mpn 128 Mpn Mpn (7229) virus, virusconfirmed type 3, and MPV; 1 TCID for positive for three viruses. Testing additional 167, 191,parainfluenza and 187) were to be positive by 50 PCR, ! Mpn! spec441 Mpn"respiratoryMpn (1837) RSV type A and parainfluenza virus type 4; 10 TCID50 for Mpn! 503 Mpn" Mpn! (286) parainfluenza virus type 2, RSV type B, and CoVs NL63 and Mpn! 549 Mpn" Mpn! (656) " ! Mpn! 566 Mpn Mpn (966) OC43; and 100 TCID50 for adenovirus, parainfluenza virus " ! Mpn! 601 Mpn Mpn (6032) type 1, and SARS-CoV. The corresponding analytical sen4/23/15 Mpn! 604 Mpn" Mpn! (3537) sitivities in genome equivalents were 50 to 250 for all virus Mpn! 119 Mpn" Mpn! (6772) types/subtypes. a Flu A, influenza A virus; Flu B, influenza B virus; Para 1, parainfluenza virus We evaluated the performance of the RVP assay by testing type 1; Para 2, parainfluenza virus type 2; Mpn, metapneumovirus. b 294 respiratory tract specimens that were submitted to the A second confirmatory PCR targeting a unique genomic region was performed to resolve the discordant results, as described in Materials and clinical virology laboratory for routine investigation of respiMethods. ratory viruses. Aliquots of each specimen were tested by rou-

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167 191 Sens) 286 62 187 108 53 349 89 443 58 128 441 503 549 566 601 604 119

Result by testa:

Does All This Stuff Actually Work?

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MAHONY

TABLE No. of specimens

123 105 5 14 47

2.

ET

AL.

Distribution of test results for

J.

DFA/culture results 294 NP specimens

DFA/culture

result

Positive Negative Positive Negative Not tested

and

test

result

Positive Negative Negative Positive Positive

Specimen no.

167 191 286 62 187 108 53 349 89 443 58 128 441 503 549 566 601 604 119

Result

DFA/culture

Flu B! Para 2! Para 2! Para 1! RSV! Flu A" Para 2" Para 2" Para 1" RSV" Flu A" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn" Mpn"

RVP

by

(MFI

MICROBIOL. RVP

testa:

reading)

Flu B" (42) Para 2" (13) Para 2" (29.5) Para 1" (20) RSV" (93) Flu A! (298) Para 2! (266) Para 2! (6447) Para 1! (215) RSV! (213) Flu A! (412) Mpn! (7229) Mpn! (1837) Mpn! (286) Mpn! (656) Mpn! (966) Mpn! (6032) Mpn! (3537) Mpn! (6772)

Second

PCRb

Flu B! Para 2! Para 2" Para 1" RSV! Flu A! Para 2! Para 2! Para 1! RSV" Flu A" Mpn! Mpn! Mpn! Mpn! Mpn! Mpn! Mpn! 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 c o n fi r m a t o r y PCR targeting a unique genomic region was performed to resolve the discordant results, as described in Materials and Methods.

indicating two false positives (numbers 286 and 62) by DFA/ culture. All of 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 eliminated 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 negatives. The sensitivity and specificity of DFA/culture were 91.9% (126/137) and 98.2% (108/110), respectively. The RVP test had a sensitivity of 97.8% (134/137) and a specificity of 96.4% (107/110). If, however, all confirmed respiratory viruses detected 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 only 126 out of 183 specimens and had a sensitivity of 68.8%. Of particular interest was the finding that 15 out of 294 (5.2%) specimens were positive for two viruses in this group of specimens. The dual infections included the following combinations: one type 1 parainfluenza virus plus one rhinovirus/ enterovirus, one type 2 parainfluenza virus plus one rhinovirus/ enterovirus, two type 3 parainfluenza viruses plus one rhinovirus/enterovirus, three RSVs plus one rhinovirus/enterovirus, one adenovirus plus one rhinovirus/enterovirus, one MPV plus one OC43 CoV, three MPVs plus one rhinovirus/ enterovirus, two OC43 CoVs plus one rhinovirus/enterovirus, and one adenovirus plus one KHU1 CoV. No specimen was positive for three respiratory viruses. Testing additional spec-

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8

CLIN.

TABLE 3. PCR results for 5 DFA/culture-positive, test-negative and 14 DFA/culture-negative, RVP test-positive specimens

RVP

RVP

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 influenza A virus subtypes H1 and H3; 0.5 TCID50 for influenza B virus, p a r a i n fl u e n z a virus type 3, and MPV; 1 TCID50 for RSV type A and p a r a i n fl u e n z a virus type 4; 10 TCID50 for p a r a i n fl u e n z a virus type 2, RSV type B, and CoVs NL63 and OC43; and 100 TCID50 for adenovirus, p a r a i n fl u e n z a virus type 1, and SARS-CoV. The corresponding analytical sensitivities 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 of respiratory viruses. Aliquots of each specimen were tested by routine 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 specimens. 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 of these, for an unadjusted sensitivity of 96.1% for the seven conventional respiratory viruses (influenza A and B viruses, 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-positive 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 of the viral genome. Table 3 shows the results for the 5 specimens that had given DFA/culture-positive, RVP test-negative discordant results and for the 14 specimens that had given DFA/culturenegative, RVP test-positive discordant results. Three of the 5 DFA/culture-positive, RVP test-negative specimens (numbers 167, 191, and 187) were confirmed to be positive by PCR,

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)

Basically an entire day Image courtesy Wikimedia commons

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Solution: Engineering Simplexa (Focus)

GeneXpert (Cepheid)

Chapter 3: Principles of Operation

Components of the FilmArray System FilmArray Pouch

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

Each FilmArray pouch is a self-contained, closed system disposable that houses all the chemistry required to isolate, amplify, and detect nucleic acid from a sample. The reservoirs in the rigid plastic component, or fitment, of the pouch (A) contain freeze-dried reagents. The flexible plastic film portion of the pouch (B) is divided into discrete segments (blisters) which, via interactions with actuators and sensors in the FilmArray Instrument, are where the following chemical processes are performed: (C) Extraction and purification of nucleic acids from a raw sample using mechanical lysis (bead beating) and magnetic bead technology (D) First-stage multiplex PCR (including reverse transcription of target RNAs) (E) Second-stage singleplex PCR and melting analysis within a multi-well array

A

Fitment and Pouch Label

B

Plastic Film Pouch

Filmarray (Biofire)

C Nucleic Acid Extraction and Purification

D Reverse Transcription and First-stage Multiplex PCR

E Second-stage Singleplex PCR

NOTE: The colored liquid in this image of a FilmArray pouch is for visualization only. FilmArray pouches do not contain colored fluid. Each pouch contains at least one internal process control. Control material is lysed and the nucleic acids of the control material are extracted along with that of the organisms contained in the sample. When the internal control is positive, proper operation of the instrument and chemical processes have been demonstrated.

FilmArray Instrument The components and operations of the FilmArray Instrument and accessories are described below. Specific step-by-step operating instructions can be found in Chapter 5 and in the Instruction Booklet provided with each FilmArray Reagent Kit or accessible via KEY-CODE access.

< 12 >

FilmArray Instrument Operator’s Manual CE IVD

2nd Generation Tests: Fully Automated

Add single buffer, put in cartridge (5 min) Load Machine (5min) PCR Run (1-2h)

2-3h

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

Basically an entire day

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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

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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?

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4-day stock-out of Xpert cartridges occurred during the study, which affected 6 specimens from 5 subjects. The median laboratory processing duration of Xpert for these specimens was 100 hours. AII Duration

When using smear microscopy for AII discontinuation decision making, the median AII duration among 201 individuals hospitalized for presumptive pulmonary tuberculosis but not diagnosed with active tuberculosis was 68.0 hours (IQR, 47.1– 97.5). The median AII duration for the Xpert AII discontinuation

effective in our population, as it would have missed 1 case of tuberculosis. Thus, having 2 specimens tested by Xpert was the most efficient strategy to determine AII discontinuation in our cohort. In October 2013, the CDC published interim practical considerations for incorporation of Xpert into diagnostic algorithms and infection control [15]. They state that for active tuberculosis evaluation and AII decision making, 3 sputum specimens should be collected 8–24 hours apart and tested by smear microscopy, a NAAT (including Xpert), or a combination of the 2 strategies [15]. Our data directly address these considerations by demonstrating that 3 specimens may not be needed and Xpert testing on 2 specimens may be the most efficient AII discontinuation strategy. Although smear microscopy

om http://cid.oxfordjournals.org/ at University of California, San Francisco on April 17, 2015

induction time was available. Failed sputum inductions contributed time data to this Kaplan-Meier curve only when the induction time was available (n = 6). Abbreviation: IQR, interquartile range.

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 PCR

190

•

CID 2014:59 (15 July)

•

Figure 3. Kaplan-Meier curve comparing airborne infection isolation duration for the Xpert MTB/RIF assay strategies on 1 specimen (n = 201), 2 specimens (n = 180), and 3 specimens (n = 148) to the smear microscopy– based strategy (n = 201). Abbreviation: IQR, interquartile range.

x

Lippincott et al Figure 2. Horizontal boxplots showing distributions of completion times for each step in sputum examination by concentrated acid-fast bacilli smear microscopy for all inpatient evaluation episodes (n = 142). Horizontal boxplots show medians and interquartile ranges (IQRs), with whisker plots displaying lower and upper adjacent values (values inside 1.5 × IQR). In addition, the median values are provided as text at the right side of the plot. Three patients were admitted twice, giving 139 patients and 142 observations.

34h PCR

65h Sput

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

Revised Device Labeling for the Cepheid Xpert

Direct Xpert Strategy

Hypothetically, if the laboratory protocol were to perform Xpert If the laboratory performed Xpert testing directly on unconcentesting using concentrated sputum at the next available standard trated sputum collected at initial evaluation in the emergency detesting time (results available 3 hours after completion of sputum partment (results available within 3 hours of arrival in the concentration), we estimated a median time from admission to laboratory), median time from admission to Xpert result would Xpert result of 34 hours (IQR, 28–53 hours; Table 4). This have been 4.5 hours (IQR, 2.9–10 hours; Table 4). This would would have reduced time spent in unnecessary respiratory isolahave reduced the time spent in unnecessary respiratory isolation tion by a median of 35 hours (IQR, 24–36 hours). Aggregated by a median of 45 hours (IQR, 35–46 hours). Aggregated across across all 133 patients with negative tuberculosis cultures, this all 133 patients with negative tuberculosis cultures, this would MTB/RIF Assa... https://vpn.ucsf.edu/mmwr/preview/mmwrhtml/,DanaInfo=ww... would have saved a total of 159 days (95% CI, 75–242 days) of have saved a total of 258 days (95% CI, 227–288 days) of unnecunnecessary respiratory isolation during the 1-year study period. essary respiratory isolation during the 1-year study period.

Table 4. Hypothetical Impact of Xpert Assay on Time to Testing Completion and Duration of Respiratory Isolation Among Patients With Negative Tuberculosis Cultures (n = 133)

Morbidity and Mortality Weekly Report (MMWR)

Smear Microscopy Strategya

Impact Time to result, h, median (IQR)

Concentrated Xpert Strategyb

Direct Xpert Strategyc

34 (28–53)

4.5 (2.9–10)

159 (75–242)

258 (227–288)

66 (58–85)

Revised Device LabelingTime for the Cepheid Xpert MTB/RIF Assay35for ... (24–36)Detecting 45 (35–46) savings vs microscopy, h, median (IQR) Total time in isolation, days/y (95% CI) tuberculosis 840 (116–1564) 684 (0–1410) 35 (31–39) Mycobacterium d

Total time savings vs control, days/y (95% CI)

...

Weekly

Abbreviations: AFB, acid-fast bacilli; CI, confidence interval; IQR, interquartile range; smear, AFB smear microscopy; Xpert, GeneXpert MTB/RIF assay.

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

b

Collection of 1 sputum sample for processing using NALC-NaOH concentration and testing by Xpert.

c

Collection of 1 sputum sample for direct testing by Xpert.

a

Collection of 2 sputum samples on separate days for N-acetyl-L-cysteine–sodium hydroxide (NALC-NaOH) concentrated acid-fast bacilli smear microscopy.

d

reflect within-patient differences and are not equal toHealth, differences between medians each strategy. Division of Microbiology Devices, Office of InSavings Vitro Diagnostics and Radiological Center for for Devices and Radiological Health, Food and Drug Administration

“…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 sputum warranted…” When compared with the results of two or three serial fluorescent-stained acid-fast smears, a single Xpert MTB/RIF Assay

Xpert for Isolation Room Triage • with CID 2014:59 (15 November) • The Food and Drug Administration (FDA) has cleared the Xpert MTB/RIF Assay (Cepheid; Sunnyvale, California) 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 for patients with suspected pulmonary tuberculosis (1). This change reflects the outcome of a recent multicenter international study demonstrating that negative Xpert MTB/RIF Assay results from either one or two sputum specimens are highly predictive of the results of two or three negative acid-fast sputum smears.*

result detected approximately 97% of patients who were acid-fast bacilli (AFB) smear–positive and culture-confirmed as infected with Mycobacterium tuberculosis complex (MTBC), and two serial Xpert MTB/RIF Assay results detected 100% of AFB smear– positive/MTBC culture-positive patients. In the setting of an overall prevalence of culture-confirmed pulmonary tuberculosis of 22.4% (14.2% [88 of 618] in the United States and 37.1% [127 of 342] outside the United States), a single negative Xpert MTB/RIF Assay result predicted the absence of AFB smear–positive pulmonary tuberculosis with a negative predictive value of 99.7% (99.6%% in the United States and 100% outside the United States); for two serial negative Xpert MTB/RIF Assay results, the negative predictive value was 100%. These findings confirm the results from earlier reports (2,3). In addition, one or two Xpert MTB/RIF Assay tests detected 55% and 69%, respectively, of sputum specimens that were AFB smear–negative but culture-positive for MTBC. Updated labeling for the Xpert MTB/RIF Assay includes the recommendation that the decision whether to test one or two sputum specimens in determining the need for continued AII should be based on specific clinical circumstances and institutional guidelines. Clinical decisions regarding the need for continued AII should always occur in conjunction with other clinical and laboratory evaluations, and negative Xpert MTB/RIF Assay results should not be the sole basis for infection control practices. The revised label also includes information demonstrating that Xpert MTB/RIF Assay performance is similar in human immunodeficiency virus (HIV)infected and HIV-uninfected adults, although HIV-infected adults with pulmonary tuberculosis might be more likely to be AFB smear negative at presentation. The Xpert MTB/RIF Assay should not be used for decisions regarding the need for continued AII if MTBC has been detected by the Xpert MTB/RIF Assay or by other methods.

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Product labeling retains the recommendation that regardless of Xpert MTB/RIF Assay results, serial collection of sputum specimens for mycobacterial culture remains necessary because nucleic acid amplification testing does not detect all patients with pulmonary tuberculosis, and recovery of organisms for further characterization and drug-susceptibility testing is needed when MTBC is present. 4/23/15 Concomitant acid-fast microscopy of serial sputum specimens is also needed when excluding nontuberculosis mycobacterial disease. Readers are encouraged to review the updated product labeling and the previous related MMWR report for additional information regarding the Xpert MTB/RIF Assay (1,4). Corresponding author: Steven Gitterman, [email protected], 301-796-6694

References 1. Cepheid. Xpert MTB/RIF assay [package insert]. Sunnyvale, CA: Cepheid; 2015. Available at http://www.cepheid.com

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Downloaded from http://cid.oxfordjournals.org/ at University of California, San Francisco on April 17, 2015

142 “TB Rule-Outs”2 Kaplan-Meierw/ curveTB comparing Xpert MTB/RIF assay and 9Figure TB 2.Patients smear microscopy laboratory processing time for successfully collected re8/9specimens Sm+(nCx+ spiratory = 546). Abbreviation: IQR, interquartile range. 8/9 PCR+ Cx+

68h Sput

Case 1 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 You would: A) Admit, start Ceftriaxone, Vancomycin, +/- steroids B) Admit, start Acyclovir, request HSV PCR (2-day turnaround) C) A+B D) Send home

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

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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 S.G. Giulieri et al. Virology / Journal of S.G. Giulieri et al. / Journal of Clinical 62Clinical (2015)Virology 58–62 62 (2015) 58–62

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Table 2 Table 2 No PCR, Manual of enterovirus in the CSF on management of aseptic adult patients with aseptic Group A: not enterovirus PCR notor done/negative orculture. negative viral culture. Impact of detection Automated in the CSF on management of adult patients with meningitis. Groupmeningitis. A: enterovirus PCR done/negative negative viral Impact of detection of enterovirus NegPCR. Bact positive home-made enterovirus Group C: positive GeneXpert enterovirus assay (GXEA). Group B:real-time enterovirusreal-time PCR. Group C: positive GeneXpert enterovirus assay (GXEA). Group B: positive home-made PCR + PCR+ Group A(n = 17)

Group A(n = Group 17) B(n = 20) Group B(n = 20) Group C(n = 22) Group C(n = 22) p value

Empirical antibiotic administration (%) 11 (64) 14 (70) 12 (55) Empirical antibiotic administration (%) 11 (64) 14 (70) 12 (55) 0.6 Duration of median antibacterial 1 (0–6) 1 (0–1.9) 0.5 (0–0.5) Duration of antibacterial therapy, daystherapy, (IQR) median days (IQR) 1 (0–6) 1 (0–1.9) 0.5 (0–0.5) 0.005a Empirical acyclovir administration (%) 8 (47) 4 (20) 0 Empirical acyclovir administration (%) 8 (47) 4 (20) 0 0.001 of stay, median days (IQR) 4 (2.5–7.5) 2 (1–3.7) 2 (1–3.7) 4 (2.5–7.5) days (IQR) 0.5 (0.3–0.7) 0.5 (0.3–0.7) 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

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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

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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

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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)

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UCSF Experience

21% positive

vs

Image courtesy iyoodle.com

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21% positive

New Techniques for Unidentified Pathogens

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

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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

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Next Generation Sequencing FANCY STEP 1: Glue on artificial sequences

GO TO FANCY STEP 2

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FANCY STEP 2: Heat%DNA,%% allow%annealing%of%designed%primers%

Thermostable%polymerase%

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

1

ID Molecular Diagnostics

4/19/15

FANCY STEP 3: Sequence: Use microscope+computer to track each base added in each cluster

GET SEQUENCE OF MILLIONS OF DNA MOLECULES Ross, et al, Am J Clin Pathol 2011;136:527-539

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

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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 . 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) 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

Next Generation Sequencing: Case #3 The

n e w e ng l a n d j o u r na l

of

m e dic i n e

brief report

Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing Michael R. Wilson, M.D., Samia N. Naccache, Ph.D., Erik Samayoa, B.S., C.L.S., Samples sent (on research basis) for NGS at UCSF Mark Biagtan, M.D., Hiba Bashir, M.D., Guixia Yu, B.S., Ø  Processing was expedited given critically illB.S., patient, 3d turn-around Shahriar M. Salamat, M.D., Ph.D., Sneha Somasekar, Scot Federman, B.A., Ø  3,063,784 total sequences (mostly human) Steve Miller, M.D., Ph.D., Robert Sokolic, M.D., Elizabeth Garabedian, R.N., M.S.L.S., Fabio Candotti, Rebecca H. Buckley, M.D., Kurt D. Reed, M.D., Ø  475 sequences fromM.D., Leptospira Teresa L. Meyer, R.N., M.S., Christine M. Seroogy, M.D., Renee Galloway, M.P.H., Ø  Never seen in any other sample lab had processed Sheryl L. Henderson, M.D., Ph.D., James E. Gern, M.D., Joseph L. DeRisi, Ph.D., and Charles Y. Chiu, M.D., Ph.D. Michael R. et al NEJM 2014 ,

From the Departments of Biochemistry and Biophysics (M.R.W., J.L.D.), Neurology (M.R.W.), and Laboratory Medicine (S.N.N., E.S., G.Y., S.S., S.F., S.M., C.Y.C.), and the Department of Infec28 of Medicine, Division 4/23/15 tious Diseases (C.Y.C.), University of California, San Francisco (UCSF), and UCSF– Abbott Viral Diagnostics and Discovery Center (S.N.N., E.S., G.Y., S.S., S.F., S.M., C.Y.C.) — both in San Francisco; the Department of Medicine, Division of Allergy and Immunology (M.B., H.B., J.E.G.), and the Departments of Pathology and Labo-

SUM M A R Y

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased nextgeneration sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

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

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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!

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

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