11 Penn New Diagnostic Testing

4/23/15 1

New Diagnostic Testing in

Infectious Disease

April 23, 2015

Bennett Penn, MD/PhD [email protected]

Image courtesy Wikimedia commons

Division of Infectious Diseases

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

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

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 (?)

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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

Massive amplification: (~Trillion-fold)

Specific Sequence In middle

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

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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

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Does All This Stuff Actually Work for Detecting Respiratory Viruses?

tested, the RVP assay detected between 0.1 and 100 TCID50 ofvirus. 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 Bvirus, parainfluenza virus type 3, and MPV; 1 TCID50 forRSV type A and parainfluenza virus type 4; 10 TCID50 forparainfluenza virus type 2, RSV type B, and CoVs NL63 andOC43; and 100 TCID50 for adenovirus, parainfluenza virustype 1, and SARS-CoV. The corresponding analytical sen-sitivities in genome equivalents were 50 to 250 for all virustypes/subtypes.

We evaluated the performance of the RVP assay by testing294 respiratory tract specimens that were submitted to theclinical virology laboratory for routine investigation of respi-ratory viruses. Aliquots of each specimen were tested by rou-tine DFA plus culture, followed by the RVP test. DFA andculture were performed in the clinical virology laboratory, andthe RVP test was performed in the research laboratory bytechnologists blinded to previous results obtained for the spec-imens. For the 294 specimens, there were 228 concordantresults, including 123 positives by DFA/culture and the RVPtest and 105 negatives by both tests (Table 2). DFA/culturedetected 128 positive specimens, and the RVP test detected123 of these, for an unadjusted sensitivity of 96.1% for theseven 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 testdetected an additional 61 positive specimens, 14 of which werenegative by DFA/culture for the seven viruses tested, and 47were positive for viruses not tested for by DFA/culture. These61 additional positive specimens included 2 for influenza Avirus, 1 for parainfluenza virus type 1, 2 for parainfluenza virustype 2, 1 for parainfluenza virus type 4, 2 for RSV, 8 for MPV,39 for rhinovirus/enterovirus, 6 for OC43 CoV, 2 for NL63CoV, 1 for HKU1 CoV, and 3 specimens that were positive fortwo viruses, including 1 specimen that was positive for MPVand rhinovirus/enterovirus and 2 specimens that were positivefor OC43 and rhinovirus/enterovirus. All of the 66 specimensthat gave discordant results, including the 5 DFA/culture-pos-itive specimens that were negative by the RVP test and the 61specimens that were positive by the RVP test and negative orpositive for viruses not tested for by DFA/culture, were testedby a second PCR that targeted a different area of the viralgenome. Table 3 shows the results for the 5 specimens that hadgiven 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 of the 5DFA/culture-positive, RVP test-negative specimens (numbers167, 191, and 187) were confirmed to be positive by PCR,

indicating two false positives (numbers 286 and 62) by DFA/culture. All of the additional 61 RVP test-positive specimenswere confirmed as true positives by the second PCR. If a truepositive is defined as being positive by two or more tests (DFA,culture, the RVP test, and/or confirmatory PCR), then therewere 183 positives and 111 negatives. To determine how theRVP test performed compared to DFA and culture, we elim-inated the 47 specimens that were positive for a virus not testedfor by DFA and culture (i.e., parainfluenza 4; MPV; CoVsOC43, 229E, NL63, and HKU1; and rhinovirus/enterovirus)and used the remaining 247 specimens for analysis. Amongthese 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 hada sensitivity of 97.8% (134/137) and a specificity of 96.4%(107/110). If, however, all confirmed respiratory viruses de-tected by the RVP test are included in the analysis, then theRVP assay detected 180 out of 183 positive specimens and hadan overall sensitivity of 98.4%, whereas DFA/culture detectedonly 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 ofspecimens. The dual infections included the following combi-nations: one type 1 parainfluenza virus plus one rhinovirus/enterovirus, one type 2 parainfluenza virus plus one rhinovirus/enterovirus, two type 3 parainfluenza viruses plus onerhinovirus/enterovirus, three RSVs plus one rhinovirus/entero-virus, one adenovirus plus one rhinovirus/enterovirus, oneMPV 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 waspositive for three respiratory viruses. Testing additional spec-

TABLE 2. Distribution of DFA/culture results and RVPtest results for 294 NP specimens

No. ofspecimens DFA/culture result RVP test result

123 Positive Positive105 Negative Negative5 Positive Negative14 Negative Positive47 Not tested Positive

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

RVP test-positive specimens

Specimenno.

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 virustype 1; Para 2, parainfluenza virus type 2; Mpn, metapneumovirus.

b A second confirmatory PCR targeting a unique genomic region wasperformed to resolve the discordant results, as described in Materials andMethods.

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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?










Specimenno.

Result by testa:



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!


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"


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!




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Specimenno.

Result by testa:



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!


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"


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!




on April 18, 2015 by UC

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PCR 123/128+ (97% Sens)

Sen 69% Sens 98%

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

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

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Simplexa (Focus)

GeneXpert (Cepheid)

Filmarray (Biofire)

< 12 > FilmArray Instrument Operator’s Manual CE IVD

Chapter 3: Principles of Operation

Components of the FilmArray System

FilmArray PouchEach 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 FilmPouch

CNucleic Acid Extraction andPurification

DReverse Transcription andFirst-stage Multiplex PCR

ESecond-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.

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

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


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


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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2 Small Studies Say It Does

207 “TB Rule-Outs”1

6 TB Patients w/ TB 6/6 Sm+ Cx+ 6/6 PCR+ Cx+

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

AII DurationWhen using smear microscopy for AII discontinuation deci-sion making, the median AII duration among 201 individualshospitalized for presumptive pulmonary tuberculosis but notdiagnosed with active tuberculosis was 68.0 hours (IQR, 47.1–97.5). The median AII duration for the Xpert AII discontinuation

strategies was 20.8 hours (IQR, 16.8–32.0) for a single Xpert strat-egy (n = 201), 41.2 hours (IQR, 26.6–54.8) for a 2-specimen strat-egy (n = 180), and 54.0 hours (IQR, 43.3–80.0) for a 3-specimenstrategy (n = 148) (Figure 3). The median AII duration was lon-ger when using the smear-based strategy for decision makingcompared with any of the 3 Xpert strategies (all log-rank testsP≤ .004).

DISCUSSION

In this observational cohort study, we found that all 3 Xpert-based strategies significantly reduced AII duration comparedwith the smear microscopy–based strategy. The 3-specimenstrategy was less efficient than the 2-specimen Xpert strategy,as the median AII duration was longer (54.0 vs 41.2 hours),without any gain in tuberculosis case detection. Although faster(20.8 vs 41.2 hours), the 1-specimen Xpert strategy was lesseffective in our population, as it would have missed 1 case oftuberculosis. Thus, having 2 specimens tested by Xpert wasthe most efficient strategy to determine AII discontinuation inour cohort.

In October 2013, the CDC published interim practical con-siderations for incorporation of Xpert into diagnostic algo-rithms and infection control [15]. They state that for activetuberculosis evaluation and AII decision making, 3 sputumspecimens should be collected 8–24 hours apart and tested bysmear microscopy, a NAAT (including Xpert), or a combina-tion of the 2 strategies [15]. Our data directly address these con-siderations by demonstrating that 3 specimens may not beneeded and Xpert testing on 2 specimens may be the most effi-cient AII discontinuation strategy. Although smear microscopy

Figure 2. Kaplan-Meier curve comparing Xpert MTB/RIF assay andsmear microscopy laboratory processing time for successfully collected re-spiratory specimens (n = 546). Abbreviation: IQR, interquartile range.

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

Figure 1. Kaplan-Meier curve displaying time from airborne infectionisolation initiation to laboratory receipt of the first (n = 201), second(n = 185), and third (n = 166) respiratory specimen(s). Failed sputum induc-tions were used to determine specimen order regardless of whether aninduction time was available. Failed sputum inductions contributed timedata to this Kaplan-Meier curve only when the induction time was avail-able (n = 6). Abbreviation: IQR, interquartile range.

190 • CID 2014:59 (15 July) • Lippincott et al

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21h 68h PCR Sput

142 “TB Rule-Outs”2

9 TB Patients w/ TB 8/9 Sm+ Cx+ 8/9 PCR+ Cx+

Concentrated Xpert StrategyHypothetically, if the laboratory protocol were to perform Xperttesting using concentrated sputum at the next available standardtesting time (results available 3 hours after completion of sputumconcentration), we estimated a median time from admission toXpert result of 34 hours (IQR, 28–53 hours; Table 4). Thiswould have reduced time spent in unnecessary respiratory isola-tion by a median of 35 hours (IQR, 24–36 hours). Aggregatedacross all 133 patients with negative tuberculosis cultures, thiswould have saved a total of 159 days (95% CI, 75–242 days) ofunnecessary respiratory isolation during the 1-year study period.

Direct Xpert StrategyIf the laboratory performed Xpert testing directly on unconcen-trated sputum collected at initial evaluation in the emergency de-partment (results available within 3 hours of arrival in thelaboratory), median time from admission to Xpert result wouldhave been 4.5 hours (IQR, 2.9–10 hours; Table 4). This wouldhave reduced the time spent in unnecessary respiratory isolationby a median of 45 hours (IQR, 35–46 hours). Aggregated acrossall 133 patients with negative tuberculosis cultures, this wouldhave saved a total of 258 days (95% CI, 227–288 days) of unnec-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 WithNegative Tuberculosis Cultures (n = 133)

Impact Smear Microscopy Strategya Concentrated Xpert Strategyb Direct Xpert Strategyc

Time to result, h, median (IQR) 66 (58–85) 34 (28–53) 4.5 (2.9–10)Time savings vs microscopy, h, median (IQR)d . . . 35 (24–36) 45 (35–46)Total time in isolation, days/y (95% CI) 840 (116–1564) 684 (0–1410) 35 (31–39)Total time savings vs control, days/y (95% CI) . . . 159 (75–242) 258 (227–288)

Abbreviations: AFB, acid-fast bacilli; CI, confidence interval; IQR, interquartile range; smear, AFB smear microscopy; Xpert, GeneXpert MTB/RIF assay.a Collection of 2 sputum samples on separate days for N-acetyl-L-cysteine–sodium hydroxide (NALC-NaOH) concentrated acid-fast bacilli smear microscopy.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.d Savings reflect within-patient differences and are not equal to differences between medians for each strategy.

Figure 2. Horizontal boxplots showing distributions of completion times for each step in sputum examination by concentrated acid-fast bacilli smearmicroscopy for all inpatient evaluation episodes (n = 142). Horizontal boxplots show medians and interquartile ranges (IQRs), with whisker plots displayinglower 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 patientswere admitted twice, giving 139 patients and 142 observations.

Xpert for Isolation Room Triage • CID 2014:59 (15 November) • 1357

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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 DetectingMycobacterium tuberculosis

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

Division of Microbiology Devices, Office of In Vitro Diagnostics and Radiological Health, Center 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 expandedintended use that includes testing of either one or two sputum specimens as an alternative to examination of serial acid-fast stainedsputum smears to aid in the decision of whether continued airborne infection isolation (AII) is warranted for patients with suspectedpulmonary tuberculosis (1). This change reflects the outcome of a recent multicenter international study demonstrating that negativeXpert MTB/RIF Assay results from either one or two sputum specimens are highly predictive of the results of two or three negativeacid-fast sputum smears.*

When compared with the results of two or three serial fluorescent-stained acid-fast sputum smears, a single Xpert MTB/RIF Assayresult detected approximately 97% of patients who were acid-fast bacilli (AFB) smear–positive and culture-confirmed as infected withMycobacterium 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 of22.4% (14.2% [88 of 618] in the United States and 37.1% [127 of 342] outside the United States), a single negative Xpert MTB/RIFAssay 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 negativepredictive value was 100%. These findings confirm the results from earlier reports (2,3). In addition, one or two Xpert MTB/RIFAssay 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 sputumspecimens 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 laboratoryevaluations, and negative Xpert MTB/RIF Assay results should not be the sole basis for infection control practices. The revised labelalso 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 smearnegative at presentation. The Xpert MTB/RIF Assay should not be used for decisions regarding the need for continued AII if MTBChas been detected by the Xpert MTB/RIF Assay or by other methods.

Product labeling retains the recommendation that regardless of Xpert MTB/RIF Assay results, serial collection of sputum specimensfor mycobacterial culture remains necessary because nucleic acid amplification testing does not detect all patients with pulmonarytuberculosis, and recovery of organisms for further characterization and drug-susceptibility testing is needed when MTBC is present.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 informationregarding the Xpert MTB/RIF Assay (1,4).

Corresponding author: Steven Gitterman, [email protected], 301-796-6694

References

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

1.

Chaisson LH, Roemer M, Cantu D, et al. Impact of GeneXpert MTB/RIF Assay on triage of respiratory isolation rooms forinpatients with presumed tuberculosis: a hypothetical trial. Clin Infect Dis 2014;59:1353–60.

2.

Lippincott CK, Miller MB, Popowitch EB, et al. Xpert MTB/RIF Assay shortens airborne isolation for hospitalized patients withpresumptive tuberculosis in the United States. Clin Infect Dis 2014;59:186–92.

3.

CDC. Availability of an assay for detecting Mycobacterium tuberculosis, including rifampin-resistant strains, andconsiderations for its use—United States, 2013. MMWR Morb Mortal Wkly Rep 2013;62:821–4.

4.

* These results represent independent FDA analysis of results from study ACTG A5295/TBTC 34. Additional information is availablefrom Luetkemeyer A, Firnhaber C, Kendall M, et al., on behalf of the ACTG A5295/TBTC 34 study teams. Xpert MTB/RIF versus AFB

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You would:

A) Admit, start Ceftriaxone, Vancomycin, +/- steroids


C) A+B

D) Send home



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

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+

S.G. Giulieri et al. / Journal of Clinical Virology 62 (2015) 58–62 61

Table 2Impact of detection of enterovirus in the CSF on management of adult patients with aseptic meningitis. Group A: enterovirus PCR not done/negative or negative viral culture.Group B: positive home-made real-time enterovirus PCR. Group C: positive GeneXpert enterovirus assay (GXEA).

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

Empirical antibiotic administration (%) 11 (64) 14 (70) 12 (55) 0.6Duration of antibacterial therapy, 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 0.001Length of stay, median days (IQR) 4 (2.5–7.5) 2 (1–3.7) 0.5 (0.3–0.7) <0.0001b

Hospitalization costs, median, $ (IQR) 5458 (2676–6274) 2796 (2062–5726) 921 (765–1230) <0.0001c

IQR = interquartile range.a Group A vs. B: p = 1.0; group A vs. C: p = 0.01*; group B vs. C: p = 0.03.b Group A vs. B: p = 0.3; group A vs. C: p < 0.0001*; group B vs. C: p < 0.0001*.c Group A vs. B: p = 1.0; group A vs. C: p < 0.0001*; group B vs. C: p < 0.0001*.

Fig. 1. Impact of enterovirus detection in CSF on duration of antibacterial therapy (Panel A) and length of hospital stay (Panel B) in aseptic meningitis. Group A: enterovirusPCR not done/negative or negative viral culture. Group B: positive home-made real-time enterovirus PCR. Group C: positive GeneXpert enterovirus assay (GXEA). In Panel B,an outlier in group B (25.5 days) is not shown.

laboratories, while in the majority of facilities analyses need to bebatched in one single daily analytical run on working days.

GXEA is characterized by 82–100% sensitivity, 100% specificity,100% positive predictive value, and 96–100% negative predic-tive value, when compared with home-made real-time PCR asgold standard [24,25]. In 434 CSF specimens, GXEA had 95% sen-sitivity and 100% specificity vs. the combination of real-timePCR and viral culture [12]. As prompt termination of empiricalantibacterial treatment and patient discharge are main aims of EVdetection, specificity and positive predictive value are key require-ments.

EV and bacterial coinfection may theoretically interfere with theclinical application of a positive GXEA result. However, coinfectionhas been only anecdotally observed in children [26,27]: a large mul-ticenter study reported no bacterial co-infection in 735 childrenwith PCR-documented EV meningitis [28].

Limitations of this study are its small sample size and obser-vational design comparing patients with positive GXEA resultswith those with and without documented EV AM from an exist-ing meningitis cohort. In addition to rapid EV detection by GXEA,improvement over the study period in diagnostic and anti-infectivemanagement of meningitis may have contributed to the presentfindings. Nevertheless, patients from groups B and C were verysimilar in terms of clinical characteristics and management wasperformed according to the same diagnostic and therapeuticalgorithm. To highlight the clinical implications of documentedEV etiology, a control group of AM without documented causewas studied. Its heterogeneity represents however a limitation:suspected culture-negative bacterial meningitis after previousantibiotic therapy and/or AM due to other viruses may have influ-enced clinical decisions independently from a negative or missingEV detection. As a matter of fact, this group exhibited a wider

Fig. 2. Correlation between turn-around time of molecular assays and hospitalization costs. Panel A: group B, i.e. patients with positive home-made real-time enterovirusPCR. Panel B: group C, i.e. patients with positive GeneXpert enterovirus assay (GXEA). The relationship between turn-around time and hospitalization was analyzed bySpearman’s correlation and linear regression.

S.G. Giulieri et al. / Journal of Clinical Virology 62 (2015) 58–62 61

Table 2Impact of detection of enterovirus in the CSF on management of adult patients with aseptic meningitis. Group A: enterovirus PCR not done/negative or negative viral culture.Group B: positive home-made real-time enterovirus PCR. Group C: positive GeneXpert enterovirus assay (GXEA).

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

Empirical antibiotic administration (%) 11 (64) 14 (70) 12 (55) 0.6Duration of antibacterial therapy, 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 0.001Length of stay, median days (IQR) 4 (2.5–7.5) 2 (1–3.7) 0.5 (0.3–0.7) <0.0001b

Hospitalization costs, median, $ (IQR) 5458 (2676–6274) 2796 (2062–5726) 921 (765–1230) <0.0001c

IQR = interquartile range.a Group A vs. B: p = 1.0; group A vs. C: p = 0.01*; group B vs. C: p = 0.03.b Group A vs. B: p = 0.3; group A vs. C: p < 0.0001*; group B vs. C: p < 0.0001*.c Group A vs. B: p = 1.0; group A vs. C: p < 0.0001*; group B vs. C: p < 0.0001*.

Fig. 1. Impact of enterovirus detection in CSF on duration of antibacterial therapy (Panel A) and length of hospital stay (Panel B) in aseptic meningitis. Group A: enterovirusPCR not done/negative or negative viral culture. Group B: positive home-made real-time enterovirus PCR. Group C: positive GeneXpert enterovirus assay (GXEA). In Panel B,an outlier in group B (25.5 days) is not shown.

laboratories, while in the majority of facilities analyses need to bebatched in one single daily analytical run on working days.

GXEA is characterized by 82–100% sensitivity, 100% specificity,100% positive predictive value, and 96–100% negative predic-tive value, when compared with home-made real-time PCR asgold standard [24,25]. In 434 CSF specimens, GXEA had 95% sen-sitivity and 100% specificity vs. the combination of real-timePCR and viral culture [12]. As prompt termination of empiricalantibacterial treatment and patient discharge are main aims of EVdetection, specificity and positive predictive value are key require-ments.

EV and bacterial coinfection may theoretically interfere with theclinical application of a positive GXEA result. However, coinfectionhas been only anecdotally observed in children [26,27]: a large mul-ticenter study reported no bacterial co-infection in 735 childrenwith PCR-documented EV meningitis [28].

Limitations of this study are its small sample size and obser-vational design comparing patients with positive GXEA resultswith those with and without documented EV AM from an exist-ing meningitis cohort. In addition to rapid EV detection by GXEA,improvement over the study period in diagnostic and anti-infectivemanagement of meningitis may have contributed to the presentfindings. Nevertheless, patients from groups B and C were verysimilar in terms of clinical characteristics and management wasperformed according to the same diagnostic and therapeuticalgorithm. To highlight the clinical implications of documentedEV etiology, a control group of AM without documented causewas studied. Its heterogeneity represents however a limitation:suspected culture-negative bacterial meningitis after previousantibiotic therapy and/or AM due to other viruses may have influ-enced clinical decisions independently from a negative or missingEV detection. As a matter of fact, this group exhibited a wider

Fig. 2. Correlation between turn-around time of molecular assays and hospitalization costs. Panel A: group B, i.e. patients with positive home-made real-time enterovirusPCR. Panel B: group C, i.e. patients with positive GeneXpert enterovirus assay (GXEA). The relationship between turn-around time and hospitalization was analyzed bySpearman’s correlation and linear regression.

4d/ $5,500

2d/ $2700

12h/ $950

Giulier et al J Clin Virology 2015

4/23/15 16

You would:

A) Admit, start Ceftriaxone, Vancomycin, +/- steroids


C) A+B

D) Send home

E) Get Enterovirus PCR, send home if +



Case 1

Outcome measures were assessed between variousgroups in the data set. Groups compared were (1) thosetested before and after RRP implementation regardless ofwhether the test result was positive or negative, (2) thosetested before and after RRP implementation based onpositive or negative result, and (3) those tested after RRPimplementation only.

Pre-RRP and Post-RRP Groups Regardless of Whetherthe Test Result Was Positive or Negative

The mean time to the test result was 1119 minutes (range,250–3705 minutes) in the pre-RRP group compared with383 minutes (range, 72–3143 minutes) in the post-RRPgroup (P , .001) (Table 4). The LOS in the ED increased by26 minutes in the post-RRP group (P ¼ .002), and thepercentage of patients who received PCR results in the EDbefore admission increased from 13.4% (n¼ 49) in the pre-RRP group to 51.6% (n¼ 398) in the post-RRP group (P ,.001). The number of patients receiving antibiotics and theinpatient LOS did not differ in the 2 groups. However, theduration of antibiotic use decreased for patients in the post-RRP group by 0.4 day (P ¼ .003). There were no deaths oradmissions to the ICU in either group.

The analysis above was repeated with patients who werepositive only for RSV, which provides a homogeneouspopulation to analyze with respect to the respiratorypathogen and, presumably, illness. Results were similar,with the post-RRP group showing a shorter test turnaroundtime (P , .001), more patients with a result in the ED (P ,.001), a longer ED stay (P¼ .01), and a decreased duration ofantibiotic use by 0.4 day (P ¼ .02).

Pre-RRP and Post-RRP Groups, With Analysis of PatientsBased on Positive or Negative Result

Table 5 compares the pre-RRP and post-RRP groups inlight of whether the test result was negative or positive. Thedecreased time to the test result, the increase in the

percentage of patients receiving PCR results before admis-sion, and the increased ED LOS remained significantregardless of the viral result being positive or negative.The inpatient LOS was shorter for the group with a positiveviral test result following implementation of the RRP (3.5days pre-RRP versus 3.2 days post-RRP, P ¼ .03). Incomparison, there was no difference in the inpatient LOSfor patients with a negative result regardless of whether thetest was before or after RRP implementation (P ¼ .88). Inaddition, patients with a positive viral test result in the post-RRP group were prescribed antibiotics for less time (3.2 dayspre-RRP versus 2.7 days post-RRP, P , .001) and were inisolation for a shorter period (82 hours pre-RRP versus 75hours post-RRP, P ¼ .03) than patients tested before RRPimplementation. These differences were not seen withpatients who had a negative result. Because of the possibilitythat the absence of influenza before RRP implementationcould confound the analysis, we performed a subanalysisthat removed patients with influenza. The results weresimilar. Patients who were positive for viruses other thaninfluenza before and after RRP implementation showedsignificantly decreased time to the test result, more PCRresults received in the ED, and shorter duration of antibioticuse compared with patients who were viral negative. Theinpatient LOS and the time in isolation were both decreasedin patients having a positive result compared with patientshaving a negative result, but statistical significance was notmaintained.

Post-RRP Group, With Analysis Based on the Timeto a Positive Test Result

Given that the test turnaround time was variable in thepost-RRP group, the inpatient LOS and the duration ofantibiotic use were also evaluated based on the length oftime it took to provide a test result. Patients having therespiratory panel results reported in less than 4 hours werecompared with patients having the respiratory panel results

Table 4. Outcomes Before and After Rapid Respiratory Panel (RRP) Implementation Regardless of Whether the TestResult 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) ,.001PCR results received in ED before admission, No. (%) 49 (13.4) 398 (51.6) ,.001Antibiotic prescribed, No. (%) 268 (73.4) 555 (72.0) .61Antibiotic use, mean (SD), d 3.2 (1.6) 2.8 (1.6) .003Inpatient LOS, mean (SD), d 3.4 (1.7) 3.2 (1.6) .16ED LOS, mean (SD), min 256 (97) 282 (115) .002Time in isolation, mean (SD), h 73 (41) 70 (38) .27

Abbreviations: ED, emergency department; LOS, length of stay; PCR, polymerase chain reaction.

Table 5. Outcomes Before and After Rapid Respiratory Panel (RRP) Implementation Based on Whether the Test ResultWas Positive or Negative

Variable

Viral Negative

P Value

Viral Positive

P ValuePre-RRP

(n ¼ 145)Post-RRP(n ¼ 169)

Pre-RRP(n ¼ 216)

Post-RRP(n ¼ 597)

Time to test result, mean (SD), min 1129 (511) 377 (275) ,.001 1113 (482) 385 (298) ,.001PCR results received in ED before admission, No. (%) 26 (17.9) 89 (52.7) ,.001 23 (10.7) 309 (51.8) ,.001ED LOS, mean (SD), min 248 (232) 277 (122) .03 262 (98) 284 (113) .02Antibiotic prescribed, No. (%) 109 (75.2) 136 (80.5) .26 157 (72.7) 416 (69.7) .41Antibiotic use, mean (SD), d 3.1 (1.6) 3.1 (1.7) .99 3.2 (1.6) 2.7 (1.5) ,.001Inpatient LOS, mean (SD), d 3.2 (1.6) 3.2 (1.6) .88 3.5 (1.8) 3.2 (1.6) .03Time in isolation, mean (SD), h 60 (36) 43 (36) .13 82 (43) 74 (38) .03

Abbreviations: ED, emergency department; LOS, length of stay; PCR, polymerase chain reaction.

4 Arch Pathol Lab Med Impact of a Rapid Respiratory Panel Test on Patient Outcomes—Rogers et al

Example #3: Respiratory Viruses






B Plastic FilmPouch







Rogers et al Arch Path Lab Med 2014

1st Gen 2nd Gen 50% of Results While Patient is in ER

4/23/15 17






B Plastic FilmPouch







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.

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


4/23/15 19

16s PCR: Principle

Sequence to get molecular fingerprint of the pathogen

Sequence every single bacteria has

Unique sequences each species

Thermostable,polymerase,

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

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.


-Started on RIPE for probable TB

4/23/15 21


-Started on RIPE for probable TB -Portion of formalin-fixed path tissue sent to CA DPH for TB PCR -> NEGATIVE


-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

4/23/15 22




-> + M. Avium Complex (MAC)




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

4/23/15 23


Pt now ~2 months into treatment, feeling much better, BMT pending

Several Laboratories offer 16s PCR

46

University of Washington Mayo Clinic Harvard

4/23/15 24

Several Laboratories offer 16s PCR

47

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

48

21% positive

Image courtesy iyoodle.com

21% positive

vs

4/23/15 25

New Techniques for Unidentified Pathogens

49


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

4/23/15 26

Next Generation Sequencing

FANCY STEP 1:

51

Glue on artificial sequences

GO TO FANCY STEP 2

FANCY STEP 2:

52

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

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

4/19/15 ID Molecular Diagnostics

1

Heat%DNA,%%allow%annealing%of%designed%primers%

Thermostable%polymerase%

FANCY STEP 3:

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

GET SEQUENCE OF MILLIONS OF DNA MOLECULES

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)

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

T h e n e w e ngl a nd j o u r na l o f m e dic i n e

n engl j med 370;25 nejm.org june 19, 20142408

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., Mark Biagtan, M.D., Hiba Bashir, M.D., Guixia Yu, B.S.,

Shahriar M. Salamat, M.D., Ph.D., Sneha Somasekar, B.S., Scot Federman, B.A., Steve Miller, M.D., Ph.D., Robert Sokolic, M.D., Elizabeth Garabedian, R.N., M.S.L.S.,

Fabio Candotti, M.D., Rebecca H. Buckley, M.D., Kurt D. Reed, M.D., Teresa L. Meyer, R.N., M.S., Christine M. Seroogy, M.D., Renee Galloway, M.P.H., 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.

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 Medicine, Division of Infec-tious Diseases (C.Y.C.), University of Cali-fornia, 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 De-partment of Medicine, Division of Allergy and Immunology (M.B., H.B., J.E.G.), and the Departments of Pathology and Labo-ratory Medicine (S.M.S., K.D.R.) and Pe-diatrics (T.L.M., C.M.S., S.L.H., J.E.G.), University of Wisconsin, Madison; the Experimental Transplantation and Immu-nology Branch, Center for Cancer Research, National Cancer Institute, National Insti-tutes of Health, Bethesda, MD (R.S., E.G., F.C.); the Departments of Pediatrics and Immunology, Division of Allergy and Im-munology, Duke University, Durham, NC (R.H.B.); and the Centers for Disease Con-trol and Prevention, Atlanta (R.G.). Address reprint requests to Dr. Chiu at the De-partment of Laboratory Medicine, Univer-sity of California, San Francisco, 185 Berry St., Box 134, San Francisco, CA 94107, or at [email protected].

This article was published on June 4, 2014, at NEJM.org.

N Engl J Med 2014;370:2408-17.DOI: 10.1056/NEJMoa1401268Copyright © 2014 Massachusetts Medical Society.

SUMM 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 pro-gressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation 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.

More than half the cases of meningoencephalitis remain un-diagnosed, despite extensive clinical laboratory testing.1-4 Because more than 100 different infectious agents can cause encephalitis, establishing

a diagnosis with the use of cultures, serologic tests, and pathogen-specific PCR assays can be difficult. Unbiased next-generation sequencing has the potential to revolutionize our ability to discover emerging pathogens, especially newly identified viruses.5-8 However, the usefulness of next-generation sequencing for the diagnosis of infectious diseases in a clinically relevant timeframe is largely unexplored.9 We used unbiased next-generation sequencing to identify a treatable, albeit rare, bacte-rial cause of meningoencephalitis. In this case, the results of next-generation se-quencing contributed directly to a dramatic effect on the patient’s care, resulting ultimately in a favorable outcome.

C A SE R EPORT

A 14-year-old boy with severe combined immunodeficiency (SCID) caused by aden-osine deaminase deficiency and partial immune reconstitution after he had under-gone two haploidentical bone marrow transplantations initially presented to the emergency department in early April 2013 after having had headache and fevers, with temperatures up to 39.4°C, for 6 days (Fig. 1A). He was admitted to the hospi-

The New England Journal of Medicine Downloaded from nejm.org at UC SHARED JOURNAL COLLECTION on April 14, 2015. For personal use only. No other uses without permission.

Copyright © 2014 Massachusetts Medical Society. All rights reserved.

Michael R. et al NEJM 2014 ,

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

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!

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

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11 Penn New Diagnostic Testing