Presented at the 113th Annual Meeting of the American Society for Microbiology

CONTACT INFORMATIONMarc Niebelmarcniebel@nhs.net

See all of BioFire’s scientific posters by scanning the QR code to access the Scientific Poster Page

Niebel, M.a, Bourzac, K.M.b, Jones, B.a, Alexander C.L.a

aScottish Parasite Diagnostic and Reference Laboratory, Stobhill Hospital, Glasgow. bBioFire Diagnostics, Inc. Salt Lake City, UT.

INTRODUCTIONTwo intestinal protozoan parasites, Cryptosporidium and Giardia duodenalis, are common causes

of gastrointestinal disturbances. These parasites infect the immunocompetent but those particularly

at risk are immunocompromised where severe fluid loss can be life-threatening. Routine diagnosis

of Giardia spp. is mainly through microscopic examination of stools for cysts or trophozoites.

Cryptosporidium spp. identification is performed by observing oocysts following auramine or

modified Ziehl-Neelsen staining. Further characterisation is performed in specialised reference

laboratories using molecular-based assays.

In this study, we evaluated a prototype FilmArray Gastrointestinal (GI) Panel (BioFire Diagnostics,

Inc, USA) which is an easy-to-use, rapid multiplexed nested RT-PCR platform which allows for

automated nucleic acid extraction, multiplexed dual-stage PCR, and simultaneous detection of 23

pathogens (bacteria, viruses and protozoa) from a single stool specimen in about one hour. Using

melt curve analysis, FilmArray software uses algorithms to automatically interpret the presence or

absence of multiple pathogens without additional user input.

METHODSThis study consisted of 96 residual stool specimens that were obtained from patients seeking

care for gastrointestinal illness and had been submitted to the Scottish Parasite Diagnostic and

Reference Laboratory (Glasgow, Scotland) for parasite analysis based on clinician suspicion of

parasite infection.

The specimens were initially characterised by microscopic methods. To identify Cryptosporidium

oocysts (4-6μm) faecal smears were examined using a fluorescent microscope at x200

magnification following auramine phenol staining. To identify Giardia cysts or trophozoites,

unconcentrated and concentrated wet preparations were examined under light microscopy at x10

and x40 magnification, and the dimensions of the cyst (10-12μm) recorded (Figure 1). All positive

Cryptosporidium samples and a subset of Giardia positive samples were further characterised by

parasite-specific in-house PCR assays (1, 2).

Figure 1. Auramine stained Cryptosporidium oocysts visualised with fluorescent microscopy (left) and Giardia trophozoite visualised with light microscopy (right)

All stool specimens were subsequently tested with the FilmArray GI Panel. Raw stool specimens

were stored at either room temperature or 4°C for between 1 and 498 days (median 21 days) before

testing with the FilmArray GI Panel and had not been previously frozen. In preparation for FilmAray

testing, stool was placed in Cary Blair media, then mixed with a sample lysis buffer and added

to the FilmArray GI Panel pouch. At the completion of testing, the FilmArray software performed

automated data analysis and generated a report.

CONCLUSIONTo summarise, the FilmArray GI Panel is comparable to both microscopy and in-house PCR for the detection of Giardia duodenalis and multiple Cryptosporidium spp., showing 92-100% agreement with these methods (Table 8). Additionally, the FilmArray GI Panel has the added benefit of allowing for the detection of additional GI pathogens with potential clinical implications.

Table 8. Performance Summary for FilmArray GI Panel Assays ACKNOWLEDGEMENTSDevelopment of FilmArray GI Panel was supported by NIH Grant #5R01AI089489

This poster contains information regarding assays that have not been cleared by the FDA for in vitro diagnostic use.

REFERENCES1. Caccio SM, Giacomo MD, Pozi E. Sequence analysis of the β-Giardin gene and development of a PCR-RFLP assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol 2002; 32:1023-1030.

2. Xiao L, Alderisio K, Limor J, Royer M, Lal AA. Identification of species and sources of Cryptosporidium oocysts in storm waters with small-subunit rRNA-based diagnostic and genotyping tool. Appl Environ Microbiol 2000; 66:5492–5498.

Results: Cryptosporidium Results: Co-InfectionsResults: Giardia

Bacteria• Aeromonas• Campylobacter• Clostridiumdifficile• Plesiomonas shigelloides

• Salmonella• Vibrio• Vibrio cholerae• Yersinia enterocolitica

Diarrheagenic E. coli/Shigella• Enterotoxigenic

E. coli (ETEC) lt/st• Enteropathogenic

E. coli (EPEC)• Shiga toxin producing

E. coli (STEC) stx1/stx2

• Shigella/Enteroinvasive E. coli (EIEC)

• Enteroaggregative E. coli (EAEC)

• E. coli O157

Viruses• Adenovirus F40/41• Human Astrovirus• Norovirus GI/GII

• Rotavirus A• Sapovirus

Protozoa•Cryptosporidium•Cyclospora cayetanensis

•Entamoeba histolytica•Giardia lamblia

THE FILMARRAY GI PANELSimultaneous detection of 23 targets:

Figure 2. The FilmArray System

The FilmArray is a lab-in-a-pouch medium-scale fluid manipulation system performed in a self-contained, disposable, thin-film plastic pouch. The FilmArray platform processes a single sample, from nucleic acid purification to result, in a fully automated fashion.

The FilmArray GI pouch has a fitment (B) containing all needed freeze-dried reagents and plungers that plunge liquids to the film portion of the pouch. This portion consists of stations for cell lysis (C), magnetic-bead based nucleic acid purification (D & E), first-stage multiplex PCR (F & G) and an array of 102, second-stage nested PCRs (I).

A. Fitment with freeze-dried reagentsB. Plungers- deliver reagents to blistersC. Sample lysis and bead collectionD. Wash stationE. Magnetic bead collection blisterF. Elution StationG. Multiplex Outer PCR blisterH. Dilution blisterI. Inner Nested PCR array

FilmArray correctly identified 26 out of 27 stool specimens that were positive by microscopy for

Cryptosporidium species, resulting in positive percent agreement (PPA) of 96.3%. Of the 69 stool

specimens that were negative by microscopy for Cryptosporidium oocysts, 68 were also negative

by FilmArray (98.6% negative percent agreement; NPA, see Table 1).

Table 1. Cryptosporidium Performance Data, Microscopy

Microscopy

Positive Negative FilmArray Performance

Film

Arr

ay

Posi

tive

26 1a 26/2796.3% PPA

Neg

ativ

e

1b 68 68/6998.6% NPA

Total 27 69a This specimen was positive for Cryptosporidium using in-house PCR (see Table 3).

bThis specimen was negative for Cryptosporidium using in-house PCR (see Table 3).

Twenty-six of the microscopy positive samples also tested positive using an in-house PCR assay

and were further characterised as C. hominis (16), C. parvum (6), C. meleagridis (1), and 3

uncharacterised (100% PPA). The sample that was identified as positive by microscopy but

negative by FilmArray was also negative using the in-house PCR assay and the specimen that

was identified as negative by microscopy but positive by FilmArray was found to be positive by

in-house PCR (1/1; 100% NPA, see Table 2 and Table 3).

Table 2. Cryptosporidium Performance Data, PCR

PCR

Positive Negative FilmArray Performance

Film

Arr

ay

Posi

tive

27 0 27/27100% PPA

Neg

ativ

e

0 1 1/1100% NPA

Total 27 1

Table 3. Investigation of Discrepant FilmArray Cryptosporidium Test Results

Sample ID FilmArray Microscopy Result PCR Final

039 Positive Negative False Pos Positive True Pos

060 Negative Positive False Neg Negative True Neg

Also of interest was FilmArray’s detection of multiple GI pathogens in stool specimens. Out of 96

specimens tested in this study, 28 (29%) were found to contain more than 1 analyte: 20 contained

2 analytes, 6 contained 3 analytes, and 2 contained 4 analytes. Examples of multiple detections

included parasite-virus (e.g. Crypto-Norovirus), parasite-E. coli (e.g. Giardia-EPEC), and Crypto-

Giardia co-infections. A complete list of specimens found to contain multiple analytes is shown in

Table 7.

Table 7. Co-infections detected with the FilmArray GI Panela

Sample ID Analytes Detected by FilmArray GI

005 Cryptosporidium, EAEC, EPEC

006 Cryptosporidium, Sapovirus

007 EAEC, EPEC, Entamoeba histolytica, Astrovirus

010 Cryptosporidium, E. coli O157

027 EAEC, Norovirus

030 Campylobacter, Cryptosporidium

034 Cryptosporidium, Giardia, Norovirus

040 Cryptosporidium, EPEC

041 Adenovirus, Cryptosporidium

044 ETEC, EAEC, EPEC

046 Giardia, Salmonella, Y. enterocolitica

048 ETEC, EPEC, Norovirus

055 EAEC, Giardia

056 Cryptosporidium, Giardia, Astrovirus, Norovirusb

057 Cryptosporidium, Norovirus

065 EPEC, Giardia

070 EPEC, Giardia

104 EAEC, Giardia

106 Giardia, Astrovirus

107 ETEC, EAEC

108 EPEC, Giardia

109 ETEC, EAEC

110 Giardia, Y. enterocolitica

115 EPEC, Giardia

108 Campylobacter, Cryptosporidium, EPEC

120 EPEC, Giardia

121 EPEC, Giardia

122 Adenovirus, Giardia

aOrganisms listed in bold font were confirmed by independent test methods

bSee Figure 3

Figure 3. DNA melt profile for specimen #056 showing multiple analytes detected (all of which were confirmed with independent PCR tests)

FilmArray correctly identified all 31 samples that were positive for Giardia cysts using microscopy

(100% PPA). Of the 65 specimens found to be negative by microscopy for Giardia, FilmArray detected

5 additional positives (92.3% NPA; Table 4).

Table 4. Giardia Performance Data, Microscopy

Microscopy

Positive Negative FilmArray Performance

Film

Arr

ay

Posi

tive

31 5a 31/31100% PPA

Neg

ativ

e

0 60 60/6592.3% NPA

Total 31 65a Follow-up testing with in-house PCR detected Giardia in 4 out of 5 specimens (see Table 6).

A subset of 15 of the microscopy-positive specimens and 5 of the microscopy-negative/FilmArray-

positive specimens were tested with an in-house PCR assay (20 total, all of which were positive for

Giardia using FilmArray). All 15 microscopy-positive specimens were also positive by PCR, along

with 4 of the 5 microscopy-negative/FilmArray-positive specimens (Table 5 and Table 6).

Table 5. Giardia Performance Data, PCR

PCR

Positive Negative FilmArray Performance

Film

Arr

ay

Posi

tive

19 1 19/19100% PPA

Neg

ativ

e

0 0 0/1N/A

Total 19 1

Table 6. Investigation of Discrepant FilmArray Giardia Test Results

Sample ID FilmArray Microscopy Result PCR Final

034 Positive Negative False Pos Positive True Pos

046 Positive Negative False Pos Negative False Pos

049 Positive Negative False Pos Positive True Pos

056 Positive Negative False Pos Positive True Pos

116 Positive Negative False Pos Positive True Pos

Evaluation of the FilmArray® Gastrointestinal Panel to Detect Giardia duodenalis and Cryptosporidium Species

OrganismMicroscopy PCR

PPA NPA PPACryptosporidium 26/27 (96.3%) 68/69 (98.6%) 26/26 (100%)

Giardia 31/31 (100%) 60/65 (92.3%) 17/17 (100%)

PCR primers are dried into the wells of the array and each primer set amplifies a unique product of the first-stage multiplex PCR. The second-stage PCR product is detected in a melting analysis using a fluorescent-double-stranded DNA binding dye, LCGreen®.