Molecular Pathology : Principles in Clinical Practice AACC

Multi-Analyte Testing of

Respiratory Viruses

Molecular Pathology : Principles in

Clinical Practice

AACC / AMP

May 8, 2012

Karen M. Frank, M.D., Ph.D.

REVISED

Speaker Disclosure Information

Grant / Research Support: bioMerieux

Learning Objectives

1. Describe different technologies used in molecular

diagnostic assays of respiratory viruses.

2. List critical factors when choosing a testing

platform for a specific institution, and compare

available commercial instruments.

3. Discuss issues that a clinician must consider

when interpreting the results of the respiratory

virus testing as a treatment plan is created.

After this presentation, you should be able to:

Outline (1) Epidemiology

(2) Molecular Testing Methodologies

(3) Controversial Issues: When to Test?

(4) Rapid Antigen Assays

(5) Example Patients and Treatment Options

(6) Infection Control

(7) Co-infection

(8) Future Issues

Epidemiology of Respiratory Syncytial Virus

• Most common cause of bronchiolitis and pneumonia in

children < 1 year of age worldwide.

• 60 million episodes & 3-4 million hospitalizations

• 66,000 – 600,000 deaths per year, 90% in developing areas.

• U.S. Data: 75,000 – 125,000 infant hospitalizations each yr.

• 400 Deaths in Infants

Villarruel et al. (2010) MMWR 59:230-233.

Nair et al. (2010) Lancet 375:1545-1555.

WHO (2009) Acute Resp. Inf. Update.

Krilov LR (2011) Expert Rev.

Infect Ther.9:27-32.

Mahony, JB (2010) Expert Rev.

Anti Infect Ther. 8:1273-1292.

Epidemiology of Influenza Virus

• In United States, 62,000 hospitalizations in 2010-

11 season.

•Range of deaths over 31 years is 3,000 to 49,000

annually.

Centers for Disease Control and Prevention

http://www.cdc.gov

• Mahony table of prevalence – make histogram with error bars

Popow and Aberle (2011) Open Micro Jnl. 5: (S2-M2) 128-134.

Regamy et al. (2008) Ped. Inf. Dis. J. 27:100-105.

Atmar et al. (2012) J. Clin. Microbiol. 50:506-508.

Mahony (2010) Expert Rev. Anti. Infect. Ther. 8:1273-1292.

Gaunt et al. (2011) J. Clin. Virol. 52:215-221.

Martin et al. (2012) Infl. Other Resp. Viruses 6:71-77.

Pavia (2011) Clin. Inf. Dis. 52(S4):S284-289.

Prevalence of Respiratory Viruses Detected during Illness

?

Disease Burden Due to Specific Viruses Varies by Age

Disease Burden Histogram Does Not Correlate with Viral Prevalence

Gaunt, E.R. et al. (2011) J. Clin. Virol. 52:215-221.

Molecular Testing Offers Significantly

Higher Assay Sensitivity

Method Sensitivity (%)

Specificity (%)

DFA or Culture

50 - >80 98 - 100

Ginocchio, G, et al. (2009) J. Clin. Virol. 45:191-195.

Leland, D. and Ginocchio, C.C. (2007) Clin. Microbiol. Rev. 20:49-78.

Liao, R. S. et al. (2009) J. Clin. Microbiol. 47:527-532.

Multiple Newly Developed Technologies

are Utilized for Multi-Analyte Testing

Caliendo, A. (2011) Clin. Inf. Dis. 52(S4):S326-S330.

Mahony, J.B. (2011) Crit. Rev. Clin. Lab. Sci. 48:217-249.

Olofsson S. et al. (2011) Expert Rev. Anti Infect. Ther. 9:615-626.

Ginocchio, C. (2011) Clin. Inf. Dis. 52(S4):S312-S325.

Endimiani, A. et al. (2011) Clin. Inf. Dis. 52(S4):S373-S383.

Gharabaghi, F. et al. (2011) Clin. Microbiol. Inf. 17:1900-1906.

Zhang, S. et al. (2011) Curr. Infect. Dis. Rep. 13:149-158.

Multiple Newly Developed Technologies are

Utilized for Multi-Analyte Testing

Multiple Newly Developed Technologies are

Utilized for Multi-Analyte Testing

http://www.luminexcorp.com

Luminex xTAG RVP

Luminex

Ginocchio, C. (2011) Clin. Inf.

Dis. 52(S4):S312-S325.

Exo=Exonuclease I

SAP=Shrimp Alkaline

Phosphatase

B=Biotin-dCTP

PE=Phycoerythrin

http://www.cepheid.com

Cepheid GenExpert

1. Integrated

Extraction

2. Real-time

Reverse Transcription

PCR.

Gen-Probe Pro-Flu + (Previously Prodesse)

http://www.cepheid.com

http://www.gen-probe.com

http://www.biomerieux-usa.com

Cepheid Smart Cycler

bioMerieux NucliSens EasyMag

1. Separate

Extraction

2. Real-time

Reverse Transcription

PCR.

Separate Assays:

ProFlu+

ProFAST

Pro hMPV+

ProAdeno+

ProParaflu+

Nanosphere Verigene RV+

http://www.nanosphere.us

1. Extraction

2. Reverse

Transcription PCR,

when needed.

3. Complementary

Oligo on Solid Array

Support.

4. Mediator Oligo on

Gold Nanoparticle.

5. Silver Amplification

of Signal.

Autogenomics Infinity RVP+

http://www.autogenomics.com

1. Integrated Extraction and Subsequent Reactions.

2. Multiplex PCR.

3. BioFilmChip Solid Microarray

Other assays:

Factor II

Factor V Leiden

Warfarin

CYP2C19

http://www.ibisbiosciences.com

Abbott PLEX-ID

1. Extraction

2. Multiplex PCR

in 96 well

plate.

3. Electrospray

Ionization

Mass

Spectrometry.

4. Multi-locus

base

composition

analysis. Multiple classes of microorganisms can be identified.

http://www.idahotech.com

Idaho Technology FilmArray

(1) Extraction of nucleic acids from sample.

(2) Nested multiplex PCR.

(3) Singleplex second-stage PCR

(4) Endpoint melting curve data.

Iquum Liat Analyzer

http://www.iquum.com

Turn-around-time of 20 minutes.

Different Sets of

Viruses are

Included in

Different

Multi-Analyte

Assays

1. Luminex xTAG RVP

2. Luminex xTAG RVP FAST

3. Gen-probe ProFlu+ (Prodesse)

4. Idaho Technologies FilmArray

5. Abbott PLEX-ID

6. Nanosphere Verigene

7. IQuum Liat Analyzer

8. Qiagen ResPlex II

9. Focus Diagnostics Simplexa

10. Autogenomics INFINITI RVP Plus

11. Seegene SeePlex

12. EraGen MultiCode PLx (Luminex)

13. PathoFinder RespiFinder

Different Sets of

Viruses are Included

in Different

Multi-Analyte Assays

Sensitivity and Specificity of Molecular Assays

is Quite High

Mahony, J.B. (2011) Crit. Rev. Clin. Lab. Sci. 48:217-249.

Gharabaghi, F. et al. (2011) Clin. Microbiol. Inf. 17:1900-1906.

Krunic, N. et al. (2011) Ann. N.Y. Acad. Sci. 1222:6-13.

Rand, K. H. et al. (2011) J. Clin. Microbiol. 49:2449-2453.

Pierce, V. M. et al. (2012) J. Clin. Microbiol. 50:364-371.

Loeffelholz, M.J. et al. (2011) J. Clin. Microbiol. 49:4083-4088.

Poritz, M. A. et al. (2011) PLoS ONE 6:e26047.

Pabbaraju, K. et al. (2011) J. Clin. Microbiol. 49:1738-1744.

Raymaekers, M. et al. (2011) J. Clin. Virol. 52:314-316.

Jannetto, R. J. et al. (2010) J. Clin. Microbiol. 48:3997-4002.

Pabbaraju, K. et al. (2008) J. Clin. Microbiol. 46:3056-3062.

Gadsby, N. J. et al. (2010) J. Clin. Microbiol. 48:2213-2216.

Arens, M. Q. et al. (2010) J. Clin. Microbiol. 48:2387-2395.

Miller, S. et al. (2010) J. Clin. Microbiol. 48:4684-4685.

Selvaraju, S. B. and Selvarangan, R. (2012) Diag. Microbiol. Inf. Dis. 72:278-281.

Balada-Llasat, J.-M. et al. (2011) J. Clin. Virol. 50:42-45.

Chen, K.-F. et al. (2011) J. Virol. Methods 173:60-66.

Hayden, R. T. et al. (2012) J. Clin. Virol. doi.10.1016/j.jcv.2011.12.020.

Caution Needed to Interpret Data Due of Reference Standard

& Number of EACH SPECIFIC VIRUS Tested

Specificity of Molecular Assays is Very High

Sensitivity of Molecular Assays is High



Sensitivity of Molecular Assays is High



How do I pick a method for MY LAB?!

1. Determine of viruses you want to detect.

2. Consider if instrument is approved.

3. Determine that matches your budget.

4. Decide level of you have.

5. Determine required.

6. EVALUATE performance: literature & colleagues.

7. Consider needs and combining assays.

Do we really need

respiratory virus

molecular testing

anyway?

Be

ds

Number of Hospital Beds Range 250 -5000

0

200

400

600

800

1000

1200

1400

1600

1800

2000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Series1

5000

Survey of Microbiology Laboratories

through Contacts on ClinMicroNet

Current Practice for Respiratory Virus Testing

Labs Offer Multiple Methods for Single Target

0

2

4

6

8

10

1 2 3 4 5

Series1

Influenza

0 1 2 3 4

0

5

10

15

1 2 3 4 5

Series1

Parainfluenza

0 1 2 3 4 0

2

4

6

8

10

12

14

1 2 3 4 5

Series1

Adenovirus

0 1 2 3 4 0

2

4

6

8

10

12

14

1 2 3 4 5

Series1

Rhinovirus/Enterovirus

0 1 2 3 4

0

2

4

6

8

10

1 2 3 4 5

Series1

RSV

0 1 2 3 4

hMPV

0

2

4

6

8

10

12

14

1 2 3 4 5

Series1

0 1 2 3 4

Number of Tests

Methods Currently in Use for Influenza Testing

0

1

2

3

4

5

6

7

8

9

10

11

12

13

1 2 3 4 5 6 7 8 9 10

Series1

Nu

mb

er

of

Lab

s

#2 Survey

CAP Proficiency Survey Shows High Use of Rapid Antigen Assays

Survey Method Virus 2009 2010 2011

VR1 Culture Influenza A Influenza B

RSV Parainfluenza

350 330 320

VR2 Direct Fluorescent

Antibody

Influenza A Influenza B

RSV Parainfluenza

290-320 240-290 240-280

VR2 Direct Fluorescent

Antibody

Metapneumo-virus

84 94

VR4 Rapid Antigen Assay

Influenza A Influenza B

RSV

1800-2800 1900-2300 1900-2300

VR4 Rapid Antigen Assay

Adenovirus 50 50-60

Survey

Method Organism 2009 2010 2011

ID2 Molecular Influenza 104 200 260

ID2 Molecular RSV 79 155 193

ID2 Molecular Parainfluenza 51 100 132

ID2 Molecular Adenovirus 70 105 133

ID2 Molecular Metapneumo-virus

53 103

ID2 Molecular Coronavirus 17 28

D Bacterial Culture E. coli/Staphyloco

ccus

2300-2800

F Mycology Culture Candida 1000

E Mycobacterial Culture

Mycobacteria 420

CAP Proficiency Survey Shows Increasing Use of Molecular Assays

Comment from Survey Participant

“Here is a Reality Check for you Karen!

Resources are limited. We function without

molecular tests.”

“I haven’t ordered a respiratory virus test in

the past 3 years.”

UCMC Primary Care Physician

Method Relative Cost To Lab

Relative Charge

to Patient

Labor Time

Labor Expertise

Rapid Antigen 1 1 Low Low

Culture 3 3 Days Medium

Direct Fluorescent

Ab

3 1.5 Hours Medium Expertise

Prodesse 7

(3 virus panel)

3 Hours High

Luminex 9

(12 virus panel)

11 Whole Shift

High

Comparison of Molecular versus Traditional Methods


http://www.cdc.gov

Rapid antigen assays for Influenza generally have

sensitivities of 40-70% (range reported is 10-80%).

“Results from these rapid tests are not accurate.”

Rapid Antigen Assays

#7 Rapid Ag Test

16 year old student with respiratory symptoms

No admission, symptoms mild

Influenza Rapid Antigen Assay

True Positive

Influenza

correctly

detected

True Negative

No influenza

Other virus likely.

False Positive

Incorrectly

diagnose

Influenza.

Likely other virus,

but not realized.

False Negative

Influenza

Not detected.

Falsely think it is

other virus.

Otherwise Healthy Person with Mild Disease

Send home

Educate on good respiratory hygiene.

Return to class based on clinical symptoms

True Positive

RSV correctly

identified.

Treat with

ribavirin.

Cohort correctly.

True Negative

Organism not

identified.

No ribavirin.

How cohort for

other viruses?

.

False Positive

Organism not

identified.

Give ribavirin.

Limit work-up.

Expose child to

RSV.

False Negative.

RSV

not detected.

Do not give

ribavirin.

Work-up ordered.

Exposing RSV-

children.

Isolation and Cohorting

2 year old child admitted

with significant with respiratory symptoms

RSV Rapid Antigen Assay

www.cdc.gov

Percent of Influenza Positive Tests is Most Often Below 40%

Prevalence Matters!!

RAPID Ag


Molecular


Off

Season

Oncologist Finds Testing Helpful

• Testing is indicated due to immune compromise from

chemotherapy, even as an outpatient, even with mild

symptoms, even outside of regular flu season.

• Therapy is affected by result

- Duration of therapy with Tamiflu

- Decision to start Ribavirin or Liposomal cidofavir

- Empiric therapy started for flu, but not for other viruses.

- In come cases, reduce or stop empiric bacterial coverage,

but often still worried about superinfection with bacteria.

Example #1 Molecular Test Useful: HIV

• 45 y/o male in respiratory distress with history of

asthma/COPD, HIV/AIDS, substance abuse, non-

adherence to his HAART therapy

• Differential DX: COPD/asthma exacerbation

Community-acquired pneumonia

Pneumocystis.

• Positive for RSV

• 31 y/o with AML-M2, status post matched-related

stem cell transplant 2 months ago, with mild graft

versus host disease and diabetes mellitus presents

with fever and cough.

• Respiratory testing positive for adenovirus

Example #2 Molecular Test Useful: Stem Cell Transplant Patient

Stem Cell Transplant Patient (cont.)

• Adenovirus PCR from the blood > 2 million copies

• Liver biopsy 5 days later: Adenovirus hepatitis with

marked hemosiderosis

• No fibrosis, Clusters of hepatocytes with some

nuclear inclusions

• Immunostain confirmed Adenovirus

• 84-year-old man with history of coronary artery disease, congestive heart failure, complete heart block, status post pacemaker placement, atrial fibrillation, numerous orthopedic issues presents with cough and fatigue.

• Positive for Influenza A

Heart condition vs infection?

Example #3 Molecular Test Useful: Heart Disease

Actions that can be taken based on test results:

(1)Stop Influenza antiviral.

(2)Start other antiviral agents.

(3)Stop some antibiotics.

(4)Cancel further diagnostic tests.

(5)Change type of isolation precautions.

Influenza Therapy Does Not Require Testing

• M2 channel inhibitors: amantidine and rimantidine

(only effective for FluA)

• Neuraminidase inhibitors – oseltamivir and zanamavir

• Should be given within first 48 h following infection

• Treatment should NOT wait for laboratory confirmation.

• Treat for 5 days, longer in some patients.

Centers for Disease Control and Prevention, http://www.cdc.gov

• Due to high levels of resistance, adamantanes not

recommended in 2010-2011 season

• Adamantane-resistant H3N2 FluA circulating since 2003

• Oseltamivir-resistant seasonal H1N1 since 2007

• Adamantane-resistent 2009 H1N1 since 2009

• Since 9/2009, 99% of flu in US is susceptible to

neuraminidase inhibitor

Influenza Treatment Depends on Resistance Patterns

When should you subtype?

MMWR (2011) 60:1

Hayden, F.G. and deJong, M.D. (2011) J. Inf. Dis. 203:6.

MMWR (2010) 59:1651

MMWR (2010) 59:901

• Low rate of resistance to oseltamivir for pandemic H1N1

• H274Y mutation in N causes resistance to oseltamivir

• S247N mutation in N causes some resistance to oseltamivir

and zanamivir.

• Dual mutation S247N/H275Y had 7000 increase in IC50 to

oseltamivir and 5 fold increase in IC50 to zanamivir.

• Other mutations: I223R in N

http://www.ecdc.europa.edu

http://www.who.int

MMWR (2009) 58:1236.

Mai, L.Q. et al. (2010) N. Engl. J. Med. 362:86-87.

Nguyen, H.T. et al. (2010) Clin. Inf. Dis. 51:983.

Flu Mutations Causing Anti-Viral Resistance

Mutations may also affect detection

RSV Treatment

Krilov, L.R. (2011) Expert Rev. Infect Ther. 9:27-32

• Ribavirin – guanosine analog

• Administered by prolonged aerosol via a small-

particle generator.

• High cost

• Cumbersome method of delivery

• Concerns about secondary exposure to caretakers

• So use is limited.

• N=603

• Children with proven RSV had 1 day shorter

hospitalization than children without a pathogen

identified.

• Fewer antibiotics 54% vs 69%

• Shorter duration of antibiotics 6.2 vs 9.3 days.

Study Demonstrating Benefit of RSV

Identification in Children

Ginocchio and colleagues: Manji, R. et al. (2009) J. Clin. Pathol. 62:998-1002.

Shadman and Wald (2011) Expert Opin Biol. Ther. 11:1455-1467.

RSV Prophylaxis Decreases Disease Burden

• Palivizumab is a humanized, murine, monoclonal antibody

that binds to RSV F protein.

• 45- 55% reduction in hospitalizations

• The 2009 Recommendations regarding Palivizumab from

the American Academy of Pediatrics attempt to balance

the cost with clinical benefit, compared to previous

recommendations.

Shadman, K.A. and Wald, E.R. (2011) Expert. Opin. Biol. Ther. 11:1455-1467.

Cox Dunn, M.C. et al. (2011) Antiviral Therapy 16:309-317.

• Leflunomide examined as a possible treatment of RSV

using rat model.

Emerging Vaccines and Drugs for RSV Treatment

Wendt, CH (1992) N. Engl. J. Med. 326:921

Nichols, W.G. (2001) Blood 98:573.

Elizaga (2001) J. Clin. Infect. Dis. 32:413

Chakrabarti, S. (2000) Clin. Infect. Dis. 31:1516.

Cobian, L. (1995) Clin. Infect. Dis. 21:1040.

Wright, J.J. (2005) J. Heart Lung Transplant 24:343.

Ison, M.G. Antivir. The.r (2007) 12:627

Alymova, I.V. (2005) Antimicrob Agents Chemother 49:398.

Alymova, I.V. (2004) Antimicrob Agents Chemother 48:1495.

Hermos, C.R. et al. (2010) Clin. Lab. Med. 30:131-148.

Wilkesmann, A. et al. (2006) Eur. J. Pediatr. 165:467-475.

Hamelin, M. et al. (2004) Clin. Inf. Dis. 38:983-990.

Wolf, D.G. et al. (2006) Pediatr. Inf. Dis. J. 25:320-324.

Beneri, C. et al. (2009) Inf. Contr. Hosp. Epidemiol. 30:1240-1241. Neofytos, D. (2007) Biol Blood Marrow Transplant 13:74.

Legrand, F. (2001) Bone Marrow Transplant 27:621

Ljungman, P. et al. (2003) Bone Marrow Transplant 31:481.

Doan, M.L. et al. (2007) J. Heart Lung Transplant 26:883.

Myers, G.D. et al. (2007) Bone Marrow Transplant 39:677.

http://clinicaltrials.gov

Limited Treatment for Respiratory Viruses

Limited Treatment for Respiratory Viruses

• Parainfluenza – Variable reports for ribavirin

• hMPV – Trial examining ribavirin

• Adenovirus – Cidofovir, Liposomal Cidofovir,

Intravenous Immune Globulin

• Oseltamivir – 600 patients

• Palivizumab – 130 patients

• Ribavirin – 50 patients

Limited Use of Antivirals at University of Chicago 2010-2011

Testing is Used for Inpatient Infection Control

Virus Status

Isolate Timing of Isolation

Influenza Positive

Contact &

Droplet

At least 7 days Longer if still symptomatic. Entire hospitalization if immunocompromised.

Influenza Negative

Contact At least 7 days Longer if still symptomatic

RSV + Contact Until 2 negative RSV tests

• All inpatients with respiratory symptoms must be

tested for influenza during influenza season

• If respiratory virus symptoms: NOT admitted to stem

cell transplant unit.

Patient/Symptoms

Admit?

Testing ?

Treat ?

Infection Control

Mild, no comorbidtiy

No No No

Mild + comorbidity

No Yes, as needed

Yes, as needed

Severe Yes Yes Yes Contact + Droplet

University Student

No No No Return after afebrile 24h off anti-pyretic

Clinical Student

No No No Afebrile 24h off anti-pyretic. Wear mask if symptoms.

Not permitted on immunosuppressived

patient units or NICU for 7 days from symptom onset

UCMC Testing Guidelines When we DON’T test.

Coinfection /Codetection

• Coinfection with influenza reported as high as 20%.

• Co-infection more frequent than would happen randomly due

to co-circulating viruses.

• Rhinovirus and Coronaviruses = most common coinfections

• 30-40% incidence of bacterial coinfection in children/infants

ventilated for RSV infection

Esper, FP et al., J. Infection (2011) doi:10.1016/j.jinf.2011.04.004

Leven, D. et al. (2010) Pediatr. Crit. Care Med. 11:390-395.

Thorburn, K. (2011) Pediatri. Crit. Care Med. 12:119

Alberle, J.H. et al. (2005) Pediatr. Inf. Dis. J. 24:605-610.

Subbarao, E.K. et al. (1989) Diagn Microbiol. Infect. Dis. 12:327-332.

Drews, A.L. et al. (1997) Clin. Infect. Dis. 25:1421-1429.

Brunstein, J.D. et al. (2008) J. Clin. Microbiol. 46:97-102.

Clinical Significance of Co-infections Not Yet Clear

• Study #1 with N=229: Patients co-infected with rhinovirus

had less severe disease than those with non-rhinovirus

coinfections, without changes in influenza viral titer.

• Study #2 with N=566: Detection of multiple viruses

correlated with less severe disease.

• Study #3 with N=645: Did not confirm a significant

association between co-infection and ICU mortality.


Martin, E.T. et al. (2011) Influ. & Other Resp. Viruses

DOI:10.1111/j.1750-2659.2011.00265.x.

Martin-Loechis, I. et al. (2011) Chest 139:555-562

Further Studies are Needed to Understand Co-infection

• Some reports suggest increased morbidity with co-infection,

while other studies report no increase in morbidity.

• Not all studies group by specific viral combinations in co-

infection. This may be partially responsible for difference in

results.

• Animal models: depending on specific combination, results

showed increased severity or viral interference


How Many Viruses Should We Include?

Influenza A H1N1

Influenza A 2009 H1N1

Influenza A H3N2

Influenza B

RSV A

RSV B

Parainfluenza 1

Parainfluenza 2

Parainfluenza 3

Parainfluenza 4

Adenovirus

Metapneumovirus

Rhinovirus

Rhinovirus

Enterovirus

Coronavirus HKU1

Coronavirus NL63

Coronavirus 229E

Coronavirus OC43

Polyomavirus WU/K1

Parvovirus 4/5

Influenza C

Bocavirus

SARS Coronavirus

Influenza H5N1

CMV

HSV 27

Organisms Resistance Genes

Streptococcus pneumoniae mecA

Haemophilus influenzae blaTEM

Mycobacterium tuberculosois blaKPC

Moraxella catarrhalis blaVIM

Staphylococcus aureus blaIMP

Bordetella pertussis blaOXA

Mycoplasma pneumoniae

Chlamydophila pneumoniae

Chlamydophila psittaci

Legionella pneumophila

Pseudomonas aeruginosa

Enterobacteriaceae

Stenotrphomonas maltophilia

Acinetobacter spp.

Bacterial and Resistance Targets

to be Considered for Respiratory Disease Diagnosis

Adaped from: Tenover, F. (2011)

Clin. Inf. Dis. 52:S338-S345.

• Some studies support a correlation of more severe disease

with higher viral load, but other studies do not see a

correlation.

• Answer may be virus specific.

• Role of quantification is not yet clearly defined, particularly

for organisms that frequently colonize airways.

El Saleeby et al. (2011) J. Inf. Dis. 204:996-1002.

Utokaparch et al. (2011) Pediatr. Infect. Dis. 30:e18-e23.

Tregoning, J. S. et al. (2010) Clin. Micro. Rev. 23:74-98.

Fodha, I. et al. (2007) J. Med. Virol. 79:1951-1958.

Van Leeuwen, J.C. et al. (2012) Pediatr. Inf. Dis. J. 31:1-2.

Discrepant Result from Studies of Quantification

• Serum procalcitonin is increased with bacterial

infection and sepsis.

• Studies have identified gene expression profile

from peripheral blood to distinguish bacterial

from viral infection.

Gilbert, D.N. (2011) Clin. Inf. Dis. S346.

Amanatidou, V. et al. (2011) Crit. Rev. Immunol. 31:341-356.

Zaas et al. (2009) Cell Host Microbe 6:207-217.

Huang et al. (2011) PLoS Genet. 7:e1002234.

Biomarkers May Play a Role in Future

Diagnosis of Respiratory Infections

Severe Acute Respiratory Syndrome

•8096 cases reported

•774 deaths

•Case-fatality rate of 9.6%


Molecular Testing Capabilities in More Labs

Provides Versatility

H5N1

•502 cases

•298 deaths

•Case fatality rate 59%

•14 countries

• With 2009 H1N1, hospital labs with molecular capabilities

were able to implement new assays very rapidly, relieving

the potentially overwhelmed public health labs.

• These capabilities will likely be valuable for the next outbreak

of an emerging virus.

Summary

(1) Factors to consider when implementing molecular testing:

- Cost of instrument and reagents

- FDA approval and Reimbursement

- Data on assay performance from good studies

- Turn-around-time decisions

- Expertise required for some assays

- Rapidly changing technology (buy or lease)

- List of viral targets to be included

- Ability to run other assays on same instrument

- Computer interface for reporting results

- Ability to subtype viruses on same platform

- Likelihood that additional improvements or additional targets will be made to current platform

- Availability of validation panel

Summary

(1) Xx

(2) Molecular testing of respiratory viruses has

definitely been beneficial for patient care,

especially in immunocompromised hosts.

(3) Laboratories must be able to adapt to newly

identified viruses, changing resistance patterns,

and rapidly evolving technologies for

identification of respiratory pathogens.

Self Assessment Questions

1. Describe three molecular diagnostic methods

used for the identification of respiratory viruses.

2. List four critical factors to consider when

choosing a respiratory virus testing platform for

a specific laboratory / institution.

2. Discuss three issues that a clinician must

consider when interpreting the results of the

respiratory virus testing as a treatment plan is

created.

Thank you

[email protected]

Documents

Molecular Pathology : Principles in Clinical Practice AACC