ASSESSING THE AGREEMENT OF GRAM NEGATIVE

MICROBIAL ORGANISM IDENTIFICATION AND

RESISTANCE MARKER IDENTIFICATION BETWEEN THE

NANOSPHERE VERIGENE® SYSTEM VERSUS

STANDARD LABORATORY METHODS

Principal Investigator: Mary Jane C. Schaefer –Weg MS, MPA

Investigators: Mykola Antoschuk

Karen Erenberg, BA

Lisbeth Herrera

Brandon Petitta

Patricia Kooker, MS

Yen-Hong Kuo, PhD

Albert Rojtman, MD

Susan Sable, MT (ASCP), SM

Department: Florence M. Cook School of Medical Laboratory Science

Office of Clinical Research, Meridian Health, JSUMC Campus

Microbiology Department, Jersey Shore University Medical Center

Objectives

• State the research hypothesis

• Discuss the background information

• List the methods and study design involved

• Explain the final identification and biomarker results

• Discuss limitations and provide ideas for continued

research

Research Hypothesis

• Null Hypothesis (𝐻0):

The PCR results for gram negative identification and antibiotic

resistance testing in patient specimens will not result in the same

outcomes as current laboratory methodologies

• Alternative Hypothesis (𝐻𝐴):

The PCR results for gram negative identification and antibiotic

resistance testing in patient specimens will result in the same

outcomes as current laboratory methodologies

Current Laboratory Technology Used at JSUMC

• VITEK 2® ID/AST

– Can take 36 - 72 hours

• Nanosphere Verigene® Film Array

Polymerase Chain Reaction (PCR)

Technology:

– Only takes 2 to 4 hours

Background Information

• 2 million new cases of antibiotic resistant infections

happen every year in the United States alone

• At least 23,000 deaths occur as a result of these infections

• Patient treatment can take weeks to months – even years

Literature Review

• Evaluation of the Nanosphere Verigene Gram-Positive Blood Culture

Assay with the VersaTREK Blood Culture System and Assessment of

Possible Impact on Selected Patients

– Appropriate antibiotics given 42 hours earlier

– Population 148 patients

• Evaluation of the Nanosphere Verigene BC-GN Assay for Direct

Identification of Gram-Negative Bacilli and Antibiotic Resistance

Markers from Positive Blood Cultures and Potential Impact for More-

Rapid Antibiotic Interventions

– Medical management could have been modified 33 hours sooner

– Population 51 patients

• Our study population - 486 patients!

Research Problem

• PCR vs Current laboratory methods

– Increases speed

– Maintains accuracy

– Provides more efficient patient care

Methods – Current Lab Practices

• Blood culture bottles are incubated in the

BD FX BACTEC®

Methods – Current Lab Practices

• Gram stains are made to identify whether they are a

gram negative or gram positive organism(s)

• Positive blood culture bottles are plated on BAP, MAC

and Chocolate plates

• The physician is called with preliminary information

Methods – Current Lab Practices

• After 18 - 24 hours of growth, identification and

sensitivity studies are performed via the VITEK 2®

ID/ AST 2 Assay system

• Time to result is 42 - 48 hours

Methods - Film Array PCR

TARGETS

Acinetobacter spp.

Enterobacter spp.

Citrobacter spp.

Escherichia coli

Proteus spp

Pseudomonas aeruginosa

Klebsiella oxytoca

Klebsiella pneumoniae

RESISTANCE

KPC (carbapenemase)

NDM (carbapenemase)

CTX-M (carbapenemase)

VIM (carbapenemase)

IMP (carbapenemase)

OXA (carbapenemase)

Methods – Film Array PCR

• Gram stains are made to identify whether they are a

gram negative or gram positive organism(s)

• Bacteria is identified directly from positive culture

bottles using the Nanosphere Verigene Film Array®

system

• Time to result is 2 – 4 hours

• No need to wait for incubation

Study Design

• Retrospective investigation:– 486 patient specimen results were examined

– 16 were excluded (only VITEK 2 ID/AST results available)

• VITEK® ID/ AST 2 and Nanosphere Verigene®

results were compared and recorded– 102 specimens contained organisms that were beyond the identity

capabilities of the Film Array PCR.

– 368 specimens had at least one organism that was detected by

Film Array and Vitek 2 ID/AST

• Results were analyzed using summary statistics and

a Z-test for one proportion

Identification Results

Bacteria Nanosphere VITEK®Acinetobacter spp 5 5

Enterobacter spp. 16 16

Citrobacter spp. 3 3

E. coli 230 230

Proteus 25 25

P. aeruginosa 20 20

K. oxytoca 19 19

K. pneumonia 55 55

Other Bacteria 102 102

*Note: 4 patient samples had more than one type of Bacteria present

Identification Results

102

364

3 10

75

150

225

300

375

450

0 1 2 3

Fre

qu

en

cy

Number of Bacteria Found

Frequency of Bacteria Detection in NanosphereSpecimens

Biomarker Results

1 0 0 0 0 0 0 00 1 0 0 0 0 0 40 0 0

35

0 0 074

153

195

25 20 19

44

0

50

100

150

200

250

Acinetobacter Enterobacter Citrobacter E. coli Proteus P. aeruginosa K. oxytoca K. pneumoniae

Fre

qu

en

cy

Bacteria Detected

Biomarkers Detected by the Nanosphere by Bacterial Organism

OXA

KPC

CTX-M

None

Resistant Biomarker Results

KPC

Film Array Modified Hodge

5 4*

CTX-M

Film Array ESBL

42 42

OXA

Film Array ESBL

1 0*

No Resistant Biomarker Results

1

260

59

0

50

100

150

200

250

300

Positive Negative Not Tested

Standard Laboratory Method Results

Fre

qu

en

cy

Film Array PCR with No Resistant Markers

Results Summary

• 100% test agreement exists between both laboratory

specimen processing methods

– Bacteria Identification

– Biomarker vs. Current laboratory methods (ESBL/Hodge)

• We therefore reject the null hypothesis of non-

matching results across all samples and accept the

alternative to be true (p < 0.01)

Conclusions

• Patient specimen results shows accuracy is

maintained between methods (100% test agreement

and rejection of the null)

• Current methods take 42 - 48 hours to complete while

PCR takes 2 – 4 hours

PCR: Reliable + Fast = Better Patient Care

Study Limitations and Opportunities for Future

Research

• Limitations

– Only Gram-Negative Bacteria Considered

– Large proportion of specimens had no biomarker due to

small population size

• Further Research Opportunities:

– Perform the same type of study with Gram-Positive

Bacteria for comparison

– Consider a larger sample with more PCR biomarker

information present for further validation

References

• Beal, Stacy G. et al. “Evaluation of the Nanosphere Verigene Gram-

Positive Blood Culture Assay with the VersaTREK Blood Culture

System and Assessment of Possible Impact on Selected

Patients.” Journal of Clinical Microbiology 51.12 (2013): 3988–

3992. PMC. Web. 18 May 2015

• Hill, Joseph T. et al. “Evaluation of the Nanosphere Verigene BC-GN

Assay for Direct Identification of Gram-Negative Bacilli and Antibiotic

Resistance Markers from Positive Blood Cultures and Potential Impact

for More-Rapid Antibiotic Interventions.” Ed. R. Patel. Journal of

Clinical Microbiology 52.10 (2014): 3805–3807. PMC. Web. 18 May

2015.

Acknowledgements

• Microbiology Department - Jersey Shore University

Medical Center

• Office of Clinical Research - Jersey Shore University

Medical Center