BIOMÉRIEUX

410853 9313400 E – 2017-04

BacT/ALERT® PF Plus
INTENDED USE Additional materials required
BacT/ALERT® PF Plus Culture Bottles are used with • BacT/ALERT® Microbial Detection Systems
BacT/ALERT® Microbial Detection Systems in qualitative • Blood-drawing device
procedures for recovery and detection of aerobic and facultative • Sterile Airway Needles/Subculture Units
anaerobic microorganisms (bacteria and yeast) from blood. • Disposable gloves
• Appropriate biohazard waste containers for materials potentially
SUMMARY AND EXPLANATION contaminated with infectious agents
BacT/ALERT Microbial Detection Systems are used to determine if
Materials available from bioMérieux
microorganisms are present in blood taken from a patient
• Blood Collection Adapter Cap
suspected of having bacteremia/fungemia. The BacT/ALERT
• BacT/ALERT® Microbial Detection Systems
System and culture bottles provide both a microbial detection
• Sterile Airway Needles/Subculture Units
system and a culture medium with suitable nutritional and
environmental conditions for organisms commonly encountered in Storage instructions
blood infections. The BacT/ALERT PF Plus Culture Bottle provides BacT/ALERT PF Plus Culture Bottles are ready for use. Store in an
for detection of microorganisms when a small volume of blood is upright position protected from direct light at room temperature (15-
available. An inoculated bottle is placed into the instrument where it 30°C). An expiration date is printed on each bottle label. Do not
is incubated and continuously monitored for the presence of inoculate the culture bottles beyond the expiration date indicated. If
microorganisms that will grow in the BacT/ALERT PF Plus Culture the bottles are exposed to temperatures less than 15°C,
Bottle. precipitates may form that will disappear when the bottles are
NOTE: The information provided applies to all configurations of warmed to room temperature. Bottles must be at room temperature
BacT/ALERT Microbial Detection Systems, unless otherwise noted. before use.
Chemical or physical indications of instability
PRINCIPLE OF THE TEST Prior to use, the BacT/ALERT PF Plus Culture Bottles should be
BacT/ALERT Microbial Detection Systems utilize a colorimetric examined for evidence of damage or deterioration (discoloration).
sensor and reflected light to monitor the presence and production Bottles exhibiting evidence of damage, leakage, or deterioration
of carbon dioxide (CO2) dissolved in the culture medium. If should be discarded. The medium in undisturbed bottles should be
microorganisms are present in the test sample, carbon dioxide is clear, but there may be a slight opalescence or a trace of
produced as the organisms metabolize the substrates in the culture precipitate due to the anticoagulant SPS or the presence of
medium. When growth of the microorganisms produces CO2, the adsorbent polymeric beads; do not confuse this with turbidity
color of the gas-permeable sensor installed in the bottom of each indicative of microbial growth. Do not use a bottle which contains
1
culture bottle changes from blue-green to yellow. The lighter medium exhibiting turbidity, a yellow sensor, or excess gas
color results in an increase of reflectance units monitored by the pressure; these are signs of possible contamination.
system. Bottle reflectance is monitored and recorded by the
instrument every 10 minutes. INSTRUMENTS
Review the appropriate BacT/ALERT Microbial Detection System
REAGENTS User Manual before use.
For in vitro diagnostic use.
CAUTION: Handle specimens and inoculated culture bottles as SPECIMEN COLLECTION AND PREPARATION
though capable of transmitting infectious agents. All General Considerations
inoculated culture bottles, specimen collection needles, and 1. BacT/ALERT PF Plus Culture Bottles should be utilized by
blood-drawing devices should be decontaminated according trained healthcare personnel. Correct specimen collection is
to your institution’s procedures.
2 extremely important when obtaining blood culture specimens.
4
Refer to Cumitech 1C for the proper specimen collection
BacT/ALERT® PF Plus (color-coded yellow) – BacT/ALERT PF procedure.
Plus disposable culture bottles contain 30 mL of complex
2. Take care to prevent contamination during both bottle
medium and ≥1.6 g adsorbent polymeric beads. At time of
preparation and inoculation of the patient sample. Proper skin
manufacture, the medium consists of the following reactive
disinfection is an essential requirement to reduce the incidence
components: combination of peptones/biological extracts
of contamination.
(≥1.85% w/v), anticoagulant (≥0.083% w/v), vitamins and amino
acids (≥0.00145% w/v) carbon sources (≥0.45% w/v), trace 3. Although not recommended by bioMérieux, blood may be
elements (≥0.0005% w/v). Bottles contain an atmosphere of N2, drawn directly into collection tubes containing SPS. Tubes
O2, and CO2, under vacuum. The composition of the medium may containing other anticoagulants should never be used for
5
be adjusted to meet specific performance requirements. blood culture.
CAUTION: BacT/ALERT culture bottles contain polycarbonate. 4. bioMérieux recommends that inoculated culture bottles be
Not all disinfectants are intended for use with polycarbonate placed into the BacT/ALERT Microbial Detection System as
surfaces, and may cause bottle deterioration. Verify soon as possible after collection. If there is an unavoidable
disinfectant compatibility with polycarbonate before use on delay, inoculated bottles may be maintained at room
BacT/ALERT culture bottle surfaces. temperature up to 24 hours before loading into the instrument.
5. Optimal recovery of isolates will be achieved by adding
CAUTION: BacT/ALERT PF Plus Culture Bottles used to
maximum amounts of specimen. Use of lower volumes may
culture very small blood specimen volumes (0.5 mL or less)
adversely affect recovery and/or detection times of some
will require added blood such as sterile defibrinated horse
organisms. The bottle’s recommended specimen volume is up
blood (10.0% v/v) to support growth, particularly for the
to 4 mL and the volume collected should be monitored by
recovery of fastidious organisms such as Haemophilus
means of the 4 mL incremental markings on the bottle label.
influenzae, Streptococcus pneumoniae, and Neisseria
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gonorrhoeae.

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6. In general, culturing higher volumes of blood containing small CAUTION: Never force the syringe plunger down during
numbers of bacteria will improve recovery of bacteria by inoculation, as splashing of sample may occur. Remove
5
culture. the syringe when the fill amount is reached, as the vacuum
7. The clinical studies were conducted with blood volumes as low automatically will draw more than the recommended
as 0.1 mL. It is recommended, however, that up to 4 mL of maximum. Puncture the bottle stopper vertically to avoid
blood be inoculated in the BacT/ALERT PF Plus Culture Bottle. releasing the vacuum; a bottle without a vacuum should
To prevent over inoculation, monitor the blood volume intake not be inoculated.
into the culture bottle, using the 4 mL incremental markings on 4. Transfer the inoculated culture bottle promptly to the testing
the bottle label. laboratory.
CAUTION: Direct vacuum draw using the BacT/ALERT PF Plus
BacT/ALERT PF PLUS CULTURE BOTTLE TEST
Culture Bottle can result in the collection of greater than 4 mL
blood sample volume. As a result, caution is advised when
PROCEDURE
performing direct draw collection of blood from young Preliminary comments and precautions
children, infants, and neonates where total blood volume is a 1. Use disposable gloves and handle inoculated bottles cautiously
concern. as though capable of transmitting infectious agents. Consult a
physician immediately if contaminated materials are ingested or
Bottle preparation come in contact with open lacerations, lesions, or other breaks
1. Label the culture bottle with patient information. The icons on in skin.
the bottle label (.,#,ž) can be defined by the user.
2. Immediately clean up any spillage of contaminated material
2. Remove plastic flip-top from the culture bottle. Prior to using a 1:10 dilution of 5% sodium hypochlorite. Dispose of the
inoculation, disinfect the culture bottle top with an alcohol swab cleaning material by an acceptable method.
or equivalent. Allow to air dry.
3. All inoculated culture bottles, specimen collection needles, and
3. Clean the selected venipuncture site as recommended by your blood-drawing devices should be decontaminated according to
institution’s approved procedure. your institution’s procedures.
2

Direct draw inoculation procedure 4. These bottles should be utilized by trained healthcare
NOTE: If inoculating more than one type of BacT/ALERT blood personnel.
culture bottle using a butterfly blood collection set and direct 5. For US Only: Caution: US Federal Law restricts this device to
draw adapter cap, inoculate first the aerobic culture bottle
sale by or on the order of a licensed practitioner.
and then the adult anaerobic culture bottle so that any oxygen
trapped in the tubing will not be transferred to the anaerobic Procedural notes and precautions
bottle. 1. Great care must be taken to prevent contamination of the
patient sample during venipuncture and during inoculation into
NOTE: Monitor the direct draw process closely at all times the culture bottle since contamination could lead to a specimen
during collection to assure proper flow is obtained and to avoid being determined positive when a clinically relevant isolate is
flow of the bottle contents into the adapter tubing. Due to the not actually present.
presence of chemical additives in the culture bottle, it is
2. Obtain blood samples prior to initiating antibiotic therapy. If this
important to prevent possible backflow and subsequent
is not possible, draw blood immediately before administering
adverse reactions by following all steps below.
the next antibiotic dose.
a. Hold the culture bottle at a position below the patient’s arm
3. If inoculated culture bottles have been delayed in their receipt
with the bottle in an upright position (stopper uppermost).
into the laboratory or have been incubated prior to entry into
b. Collect the blood using a butterfly blood collection set and
the BacT/ALERT instrument, visually inspect for indications of
a blood collection adapter cap as recommended by your
microbial growth. If microbial growth is evident, treat the bottles
institution’s approved procedure and inoculate directly into
as positive and do not place in the BacT/ALERT Microbial
the culture bottle at the patient’s bedside. Although lower
Detection System for monitoring.
sample volumes can be used, recovery may be improved
using a sample volume closer to the recommended 4 mL. Laboratory procedure
c. Release the tourniquet as soon as the blood starts to flow CAUTION: General caution should be taken when subculturing
into the culture bottle, or within 2 minutes of application. positive culture bottles as they could have been overfilled or
d. Do not allow the culture bottle contents to touch the contain high gas-producing organisms. Positive culture bottle
stopper or the end of the needle during the collection contents may be under increased internal pressure. Positive
procedure. culture bottles should be transiently vented before staining or
CAUTION: A contaminated culture bottle could contain disposal to release any gas produced during microbial
positive pressure, and if used for direct draw, may cause metabolism.
reflux into the patient’s vein. Culture bottle contamination may 1. Visually inspect bottles before testing. Do not use bottles with
not be readily apparent. Monitor the direct draw process evidence of damage, leakage, or deterioration. Consider
closely to avoid reflux. Do not use a bottle that contains bottles with hemolysis, turbidity, excess gas pressure, yellow
medium exhibiting turbidity, a yellow sensor, or excess gas sensors, and/or evidence of growth as positive. Smear and
pressure; these are signs of possible contamination. subculture. Do not incubate unless smear is negative.
Syringe draw inoculation procedure 2. After culture bottles have been loaded into the instrument,
NOTE: If inoculating more than one type of BacT/ALERT blood incubate 5 days or until designated positive.
culture bottle using syringe draw, inoculate first the adult 3. Smear and subculture all positive bottles. If the smear is
anaerobic culture bottle and then the aerobic culture bottle negative, indicating a possible false positive, the bottle should
so that any oxygen trapped in the syringe will not be be reloaded into the instrument until growth of the subculture or
transferred to the adult anaerobic bottle. Line demarcations on redesignation as positive. Bottles that were initially determined
the bottle label should be used to assist in estimating the false positive and were redesignated positive should be
sample volume. smeared and subcultured.
a. Perform venipuncture and blood transfer to the 4. Negative bottles may be checked by smear and/or subculture
BacT/ALERT culture bottle according to your institution’s at some point prior to discarding as negative.
established procedures.

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5. Procedures for loading and unloading culture bottles into the 4. Infrequently, if there is a very high number of white blood cells
appropriate BacT/ALERT instrument are given in the User present in the sample, the BacT/ALERT may indicate a culture
Manual. bottle positive. In this case, the smear and subculture results
6. Do not reuse BacT/ALERT culture bottles. Dispose of may be negative.
inoculated BacT/ALERT culture bottles according to your 5. Organisms are often few in numbers and may appear
laboratory protocol. Autoclaving and/or incinerating inoculated intermittently in the blood stream; therefore, consecutive blood
2
BacT/ALERT culture bottles is appropriate. samples should be collected from each patient.
7. Utilization of coring devices (i.e., blunt needle) to puncture the 6. Promptly remove positive culture bottles when they are
septum may result in bottle leakage. signaled by BacT/ALERT to avoid possible non-viable cultures
due to autolysis or other reasons. Certain strains of
QUALITY CONTROL Streptococcus pneumoniae may be particularly prone to
A Certificate of Conformance is available for each lot of culture autolysis if they are not removed promptly after being signaled
bottles. If desired, individual laboratories can perform quality control positive.
testing of BacT/ALERT PF Plus Culture Bottles. Refer to the
7. A Gram-stained smear from a negative bottle may sometimes
appropriate BacT/ALERT User Manual and to CLSI® document
6 contain a small number of non-viable organisms that were
M22-A3.
derived from culture medium components, staining reagents,
Instrument immersion oil, or glass slides, resulting in a false positive
A BacT/ALERT Reflectance Standards kit is provided with each smear.
BacT/ALERT 3D instrument and Reflectance Calibration Standards 8. bioMérieux recommends that inoculated culture bottles be
are included with each BacT/ALERT VIRTUO instrument for the placed into the BacT/ALERT Microbial Detection System as
QC and Calibration procedures. All quality control should be part of soon as possible after collection. But, in the unavoidable cases
normal system maintenance. Refer to the appropriate BacT/ALERT when there is a delay in bottle receipt by the laboratory,
User Manual for more information. delayed entry information is provided from seeded studies in
CAUTION: If your facility’s LIS vendor sends bottle IDs and the “Performance Characteristics of the Test” section.
bottle type abbreviations to the BacT/ALERT instrument, use 9. Antimicrobial neutralization was not achieved for ceftazidime or
the correct bottle type abbreviation to avoid possible false cefepime.
positive or false negative results. For more information,
contact your local bioMérieux representative. EXPECTED VALUES
1. Percent positive cultures were observed to be 6.3% (range:
RESULTS 4.9%-8.1%) overall and 4.1% (range: 2.5%-6.4%) for significant
Positive or negative culture bottles are determined by decision- isolates from three clinical trial sites in BacT/ALERT PF Plus
making software contained in the BacT/ALERT Microbial Detection Culture Bottles that received 0.1-4 mL of blood during the
System. No action is required until the BacT/ALERT instrument BacT/ALERT PF Plus Culture Bottle clinical trial conducted with
signals a culture bottle either positive or negative. the BacT/ALERT 3D.
LIMITATIONS OF THE TEST 2. For BacT/ALERT 3D testing during the BacT/ALERT VIRTUO
Many variables involved in blood culture testing cannot be clinical trial, percent positive cultures were observed to be 1.5%
practically controlled to provide total confidence that results (range: 0.0%-11.1%) overall and 1.5% (range: 0.0%-11.1%) for
obtained are due solely to proper or improper performance of any significant isolates from three clinical trial sites in BacT/ALERT
culture medium or detection system. PF Plus Culture Bottles that received ≤4 mL of blood.
1. Patient specimens determined positive by BacT/ALERT may 3. For BacT/ALERT 3D testing during the BacT/ALERT VIRTUO
contain organisms that are positive by smear that will not grow clinical trial, percent positive cultures were observed to be 8.4%
on routine subculturing media. When this is suspected, (range: 3.6%-13.6%) overall and 6.9% (range: 3.6%-13.6%) for
specimens should be subcultured on special media. Also, significant isolates from three clinical trial sites in BacT/ALERT
BacT/ALERT positive specimens may contain organisms that FA Plus Culture Bottles that received ≤4 mL of blood.
are not seen with routine smear staining methods and may 4. For the BacT/ALERT VIRTUO clinical trial, percent positive
require both specialized staining and subculturing media for cultures were observed to be 2.0% (range: 0.0%-2.1%) overall
detection and recovery. and 1.5% (range: 0.0%-1.6%) for significant isolates from three
2. It is possible that certain rare, fastidious microorganisms will clinical trial sites in BacT/ALERT PF Plus Culture Bottles that
not grow or may grow slowly in the BacT/ALERT PF Plus received ≤4 mL of blood.
Culture Bottle growth medium. In addition, on rare occasions, 5. For the BacT/ALERT VIRTUO clinical trial, percent positive
organisms may be encountered that grow in the BacT/ALERT cultures were observed to be 8.2% (range: 6.9%-13.6%)
PF Plus Culture Bottle growth medium, but do not produce overall and 6.9% (range: 5.7%-12.5%) for significant isolates
sufficient carbon dioxide to be determined positive. If rare, from three clinical trial sites in BacT/ALERT FA Plus Culture
fastidious organisms requiring specialized media and culture Bottles that received ≤4 mL of blood.
conditions are suspected, alternative methods or extended 6. Expected percent positives will vary based on factors such as
incubation time should be considered for recovery. patient population, prevalence of significant organisms, site
3. Certain strains of Haemophilus influenzae, Neisseria location, and contamination rates. The expected values
meningitidis, and Neisseria gonorrhoeae may be sensitive to provided are based on clinical study data acquired during
the anticoagulant SPS, which may result in a lack of growth or multiple clinical studies.
low production of CO2 by these strains if an insufficient amount
of sample is inoculated into the culture bottles.

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NEUTRALIZATION OF ANTIMICROBIALS
Neutralization of antimicrobials by adsorbent polymeric beads varies depending upon dosage level and timing of specimen collection. Internal
studies tested on the BacT/ALERT 3D Microbial Detection System have demonstrated that antimicrobials are effectively neutralized by the
BacT/ALERT PF Plus medium based on 100% recovery of the organisms tested. In these tests, antimicrobials were added in clinically
relevant concentrations directly to culture bottles during inoculation with susceptible strains. The effectiveness of the antimicrobials was
confirmed by parallel testing using a non-neutralizing medium as a control. Antimicrobials from the following categories were neutralized by
the medium: penicillins, glycylcyclines, polyenes, macrolides, triazoles, echinocandins, cefazolin, cefoxitin, ceftaroline, aminoglycosides,
fluoroquinolones, lincosamides, glycopeptides, and oxazolidinones.
Antimicrobial neutralization was not achieved for ceftazidime or cefepime. Less than complete neutralization was observed for cefotaxime and
ceftriaxone. Cefotaxime was neutralized at ranges of 50% peak serum level (PSL) to 2% PSL depending on the microorganism. Ceftriaxone
was neutralized at ranges of 50% PSL to 1% PSL depending on the organism.
Antimicrobial neutralization properties are dependent upon the culture bottle material composition and are not determined by the analysis
algorithms of the BacT/ALERT Microbial Detection Systems. Representative antimicrobials selected from four of the categories listed above
were tested on the BacT/ALERT VIRTUO Microbial Detection System to confirm neutralization properties of the BacT/ALERT PF Plus Culture
Bottles. Neutralization was demonstrated for amikacin (from the aminoglycosides class), piperacillin (penicillins class), vancomycin
(glycopeptides class), and voriconazole (triazoles class). Testing demonstrated that the instrument system had no impact on culture bottle
antimicrobial neutralization properties.
For additional information on antimicrobial agents neutralized by BacT/ALERT PF Plus Culture Bottles, contact your local bioMérieux
representative.

PERFORMANCE CHARACTERISTICS OF THE TEST

BacT/ALERT 3D Microbial Detection Systems
Potentially Interfering Substances
In-house seeded studies were conducted with plasma, blood, and blood clots. Aliquots of each of these fluids also received white blood cells
at concentrations relevant to bacteremia in blood. Testing was conducted with and without microorganisms. These substances neither
interfered with recovery and detection of organisms, nor did they generate false positive results in the absence of organisms.
Analytical Sensitivity: Limit of Detection (LoD)
Data in Table 1 represent results from in-house seeded studies. A minimum of 30 replicates were tested per species. At least 95%
detection was achieved at LoD. Data in Table 1 were generated using bottles at the end of shelf life. Bottles inoculated with H. influenzae
received 4 mL pooled human blood supplementation.
Table 1. Analytical Sensitivity: Limit Of Detection (LoD)
Microorganism Strain ID LoD (CFU/bottle)
Candida albicans ATCC® 14053™ 6
Enterobacter aerogenes ATCC® 13048™ 8
Enterococcus faecalis NCTC 12697 5
Escherichia coli NCTC 12923 4
Haemophilus influenzae ATCC® 10211™ 6
Klebsiella pneumoniae STL 104016 4
Listeria monocytogenes ATCC® 15313™ 6
Pseudomonas aeruginosa NCTC 12924 4
Salmonella enterica ATCC® 14028™ 5
Staphylococcus aureus NCTC 10788 5
Streptococcus pneumoniae ATCC® 6305™ 6
NOTE: 96.7% of the bottles were subcultured within 30 minutes of being declared positive. STL 104016 was sourced from bioMérieux’s internal culture collection.

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Analytical Sensitivity: Growth Performance

Data in Table 2 represent results from in-house seeded studies with blood obtained from healthy human volunteers. Multiple strains were
tested for each species at target inoculum levels of 125 CFU/bottle. The actual inoculum levels ranged from 3-298 CFU/bottle. In this seeded
study, the BacT/ALERT PF Plus Culture Bottles were subcultured at least 24 hours after being flagged positive by the instrument. The species
listed are representatives of clinically prevalent organisms in blood cultures.
Table 2. Analytical Sensitivity: Growth Performance
Blood
Microorganism Time to Detection (hours)
% Recovery (n) Range (CFU/bottle)
Mean Range
Staphylococcus aureus 100.0 (30/30) 54-150 13.3 12.2-15.2
Escherichia coli 100.0 (30/30) 71-254 11.2 10.3-11.7
Pseudomonas aeruginosa 100.0 (12/12) 74-148 15.7 13.7-17.8
Klebsiella pneumoniae 100.0 (12/12) 89-123 11.3 10.6-12.3
Candida albicans 100.0 (30/30) 88-298 29.0 19.2-52.8
Streptococcus pneumoniae 100.0 (30/30) 3-260 13.8 10.8-16.5
Staphylococcus epidermidis 100.0 (12/12) 44-135 17.6 14.3-18.8
Enterococcus faecalis 100.0 (12/12) 63-259 11.6 11.0-12.2
Enterococcus faecium 100.0 (12/12) 25-120 12.8 11.3-14.4
Enterobacter cloacae 100.0 (12/12) 111-200 11.6 10.8-12.5
Candida glabrata 100.0 (12/12) 118-281 43.5 27.3-64.8
Haemophilus influenzae 100.0 (12/12) 105-266 14.4 12.0-16.8
Proteus mirabilis 100.0 (12/12) 36-213 12.5 11.3-14.6

Less than 100% detection was observed for some species, including Capnocytophaga ochracea, Cardiobacterium hominis, Eikenella
corrodens, Haemophilus parainfluenzae, Granulicatella adiacens, and Helicobacter cinaedi.
Clinical Study Results (Blood Cultures)
Results compare BacT/ALERT PF Plus to BacT/ALERT PF blood cultures (for all compliant pairs).
A multi-center clinical study was conducted at three different geographic sites in the U.S. comparing the performance of the BacT/ALERT PF
Plus and BacT/ALERT PF blood culture bottles for pediatric culture pairs that received blood volumes between 0.1 mL and 4 mL (compliant
pairs). A total of 2188 bottle pairs were obtained from 1086 pediatric patients suspected of blood stream bacterial/yeast infections. Subcultures
of both bottles were performed when either bottle in the set was determined to be positive by the BacT/ALERT System. A pair of bottles was
determined to have a positive status if the subculture of either the BacT/ALERT PF Plus or BacT/ALERT PF Culture Bottle was positive. A
culture bottle was determined to be a “True Positive” if the culture was flagged positive by the BacT/ALERT System and resulted in growth of
the isolate upon subculture of this bottle. True positive rates were calculated for the BacT/ALERT PF Plus and BacT/ALERT PF Culture
Bottles, and the ratio of BacT/ALERT PF Plus true positives to BacT/ALERT PF true positives was calculated to compare performance.
Clinical isolates recovered were classified as significant, contaminant, or unknown based on determination by the clinical trial sites.
A total of 172 isolates were recovered from all compliant pediatric blood culture pairs with a positive status. There were a total of 145 bottle
pairs that recovered at least one isolate by subculture of BacT/ALERT PF Plus or BacT/ALERT PF Culture Bottles. A total of 126 bottle pairs
recovered a single isolate, 12 bottle pairs recovered two isolates, six bottle pairs recovered three isolates, and one bottle pair recovered four
isolates. The total population reported in Tables 3, 4, and 5 comprises the 172 isolates recovered from positive bottle pairs and 2043 negative
bottle pairs for a total of 2215 results. The BacT/ALERT PF Plus Culture Bottle detected a total of 140 isolates compared to the BacT/ALERT
PF Culture Bottle that detected 128 isolates. Of the significant isolates, the BacT/ALERT PF Plus Culture Bottle detected a total of 91 isolates
compared to the BacT/ALERT PF Culture Bottle that detected 77 isolates. One false positive was identified by subculture of a positive
BacT/ALERT PF Plus Culture Bottle and comprised 0.05% (1/2215) of the study population.

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The following tables compare results of the BacT/ALERT PF Plus to BacT/ALERT PF blood cultures for all compliant blood culture bottles that
yielded any number of isolates on subculture (Table 3), a single isolate alone on subculture (Table 4), and multiple isolates on subculture
(Table 5).
Table 3. All Compliant Pairs With Single And Multiple Isolates Combined (Blood Cultures)
Clinical Isolate BacT/ALERT PF Plus % of BacT/ALERT PF Plus BacT/ALERT PF % of BacT/ALERT PF Ratio of True
Determination True Positives True Positives in Population True Positives True Positives in Population Positives*
Significant 91 4.1 (91/2215) 77 3.5 (77/2215) 1.182
Contaminant 24 1.1 (24/2215) 29 1.3 (29/2215) 0.828
Unknown 25 1.1 (25/2215) 22 1.0 (22/2215) 1.136
Total 140 6.3 (140/2215) 128 5.8 (128/2215) 1.094
* Ninety six (96) isolates were detected by both BacT/ALERT PF Plus and BacT/ALERT PF, 44 isolates were detected only by BacT/ALERT PF Plus, and 32
isolates were detected only by BacT/ALERT PF. The ratio of true positive rates for overall isolates was 1.094 (140/128) with a 95% CI (0.954, 1.234).7

Table 4. All Compliant Pairs With Single Isolates (Blood Cultures)

Clinical Determination BacT/ALERT PF Plus True Positives BacT/ALERT PF True Positives Ratio of True Positives*
Significant 69 61 1.131
Contaminant 17 17 1.000
Unknown 19 16 1.188
Total 105 94 1.117
* Seventy three (73) isolates were detected by both BacT/ALERT PF Plus and BacT/ALERT PF, 32 isolates were detected only by BacT/ALERT PF Plus, and 21
isolates were detected only by BacT/ALERT PF. The ratio of true positive rates for overall single isolates was 1.117 (105/94) with a 95% CI (0.957, 1.277).7

Table 5. All Compliant Pairs With Multiple Isolates (Blood Cultures)

Clinical Determination BacT/ALERT PF Plus True Positives BacT/ALERT PF True Positives Ratio of True Positives*
Significant 22 16 1.375
Contaminant 7 12 0.583
Unknown 6 6 1.000
Total 35 34 1.029
* Twenty three (23) isolates were detected by both BacT/ALERT PF Plus and BacT/ALERT PF, 12 isolates were detected only by BacT/ALERT PF Plus, and 11
isolates were detected only by BacT/ALERT PF. The ratio of true positive rates for overall multiple isolates was 1.029 (35/34) with a 95% CI (0.748, 1.310).7
In this clinical study, there were 2041 pairs of BacT/ALERT PF Plus and BacT/ALERT PF Culture Bottles with negative instrument results for
both bottles after 5 days of incubation. Among these pairs, terminal subcultures on both bottles were performed for three pairs, and no false
negative results by both BacT/ALERT PF Plus and BacT/ALERT PF Culture Bottles were observed; subculture on BacT/ALERT PF Plus
Culture Bottles alone was performed for 2034 pairs, and one false negative result was observed; both subcultures were not performed for four
pairs of bottles. Results are summarized in Table 6.
Table 6. Summary Of Percent False Negatives From Compliant Aerobic Blood Culture Pairs That Were Flagged Negative By The
Instrument For Both Bottles
Subculture Performed Subculture Performed % False Negative % False Negative
BacT/ALERT PF Plus BacT/ALERT PF BacT/ALERT PF Plus BacT/ALERT PF
Yes Yes 0.00 (0/3) 0.00 (0/3)
Yes No 0.05 (1/2034) -

Overall false negative rate for BacT/ALERT PF Plus based on a subset of terminal subcultures was 0.05% (1/2037).

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A comparative yield of microorganisms (number of isolates) recovered on subculture of BacT/ALERT PF Plus and BacT/ALERT PF cultures is
presented in Table 7.
Table 7. Comparative Yield Of Microorganisms From BacT/ALERT PF Plus And BacT/ALERT PF
BacT/ALERT BacT/ALERT PF BacT/ALERT PF
Organism Group Pediatric Subgroup BacT/ALERT PF
PF Plus Plus Fill Range (mL) Fill Range (mL)
Newborn (< 1 mo) 6 0.1-1.4 7 0.1-1.6
Infant (> 1 mo–2 yrs) 19 0.1-3.7 13 0.1-3.1
Enterobacteriaceae
Child (> 2 yrs–12 yrs) 9 0.6-3.9 7 0.3-3.2
Adolescent (>12–21 yrs) 0 1.4 1 1.6
Newborn (< 1 mo) 0 - 0 -
Fastidious (Neisseria Infant (> 1 mo–2 yrs) 1 0.2 1 0.5
meningitidis, Neisseria
sicca) Child (> 2 yrs–12 yrs) 0 - 0 -
Adolescent (>12–21 yrs) 1 1.1 1 0.6
Newborn (< 1 mo) 0 - 0 -
Yeast (Candida albicans, Infant (> 1 mo–2 yrs) 0 0.5 1 0.9
C. guilliermondii, C.
krusei, C. lusitaniae) Child (> 2 yrs–12 yrs) 5 0.9-3.7 6 1.0-3.4
Adolescent (>12–21 yrs) 1 0.2-3.0 2 2.1-2.5
Newborn (< 1 mo) 0 - 0 -
Non-fermentative Infant (> 1 mo–2 yrs) 5 1.7-3.5 6 1.5-2.5
Gram-Negative Bacilli Child (> 2 yrs–12 yrs) 2 0.9-2.2 3 1.0-2.8
Adolescent (>12–21 yrs) 0 - 0 -
Newborn (< 1 mo) 5 0.1-0.5 5 0.1-0.9
Coagulase-Negative Infant (> 1 mo–2 yrs) 12 0.1-3.0 10 0.1-3.4
Staphylococcus Child (> 2 yrs–12 yrs) 15 0.1-3.8 12 0.5-3.6
Adolescent (>12–21 yrs) 6 0.5-3.5 7 0.5-3.2
Newborn (< 1 mo) 0 0.3 1 0.1
Infant (> 1 mo–2 yrs) 5 0.5-1.5 5 0.6-1.6
Staphylococcus aureus
Child (> 2 yrs–12 yrs) 7 0.8-4.0 3 0.1-3.6
Adolescent (>12–21 yrs) 2 1.5-1.7 2 1.3-1.4
Newborn (< 1 mo) 1 0.1 1 0.1
Infant (> 1 mo–2 yrs) 9 0.2-2.9 10 0.1-3.2
Enterococcus spp.
Child (> 2 yrs–12 yrs) 2 0.2-1.0 1 0.8-1.8
Adolescent (>12–21 yrs) 8 1.5-3.1 7 1.9-2.9
Newborn (< 1 mo) 0 - 0 -
Streptococcus Infant (> 1 mo–2 yrs) 2 1.0-2.7 1 1.1-1.6
pneumoniae Child (> 2 yrs–12 yrs) 0 - 0 -
Adolescent (>12–21 yrs) 0 - 0 -
Newborn (< 1 mo) 0 - 0 -
Streptococcus spp., Infant (> 1 mo–2 yrs) 1 0.5 0 1.0
Group A, B Child (> 2 yrs–12 yrs) 0 - 0 -
Adolescent (>12–21 yrs) 0 - 0 -
Newborn (< 1 mo) 0 - 0 -
Infant (> 1 mo–2 yrs) 4 0.1-1.6 5 0.1-1.5
Other Streptococcus spp.
Child (> 2 yrs–12 yrs) 3 0.7-2.3 2 0.6-2.6
Adolescent (>12–21 yrs) 1 0.7-2.4 2 1.1-2.4
Newborn (< 1 mo) 0 - 0 -
Infant (> 1 mo–2 yrs) 1 0.6 0 0.4
Other Gram-negative*
Child (> 2 yrs–12 yrs) 0 - 0 -
Adolescent (>12–21 yrs) 0 - 0 -
Newborn (< 1 mo) 1 0.1-0.7 2 0.1-0.4
Infant (> 1 mo–2 yrs) 3 0.5-3.0 2 0.9-2.3
Other Gram-positive†
Child (> 2 yrs–12 yrs) 3 0.1-3.4 1 0.1-3.2
Adolescent (>12–21 yrs) 0 3.3 1 3.2
* Other Gram-Negative Organisms recovered in clinical study: Unidentified Gram-Negative Rods (1)
† Other Gram-Positive Organisms recovered in clinical study: Bacillus spp. (6), Corynebacterium spp. (2), Diphtheroids (1), Micrococcus spp. (2), Stomatococcus
spp. (1).
Organisms were recovered in the clinical studies at blood volumes greater than 0.1 mL. They are:
• ≥1.0 mL for Streptococcus pneumoniae
• ≥0.9 mL for non-fermenting Gram-negative rods; ≥ 0.6 mL for unidentified Gram-negative rods
• ≥0.5 mL for Group A, B Streptococcus spp.
• ≥0.3 mL for S. aureus
• ≥0.2 mL for fastidious organism (N. meningitidis and N. sicca)
• ≥0.2 mL for Yeast (Candida albicans, C. guilliermondii, C. krusei, and C. lusitaniae)

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Quality control was performed during the clinical study on each of 13 organisms (Candida albicans, Candida krusei, Enterococcus faecalis,
Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus
epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), which were
prepared using serial dilution and seeded into the BacT/ALERT PF Plus Culture Bottle at a target inoculum of 100 CFU/bottle, with an
acceptable range of 30-300 CFU/bottle. Overall quality control results were found to be acceptable. Instances where unacceptable quality
control results were observed were found to be due to technical errors (i.e., colony counts out of range, site failure to change bottle status after
positive instrument signal and positive subculture, and no supplement added). Repeat testing resulted in acceptable results.
Delayed Entry
Table 8 includes results from seeded studies using 11 species (Staphylococcus aureus, Candida albicans, Candida krusei, Escherichia coli,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Streptococcus pneumoniae, Enterococcus faecium, Haemophilus
influenzae, and Neisseria meningitidis), at target concentrations of 100 CFU/bottle (acceptable range of 30-300 CFU/bottle) were generated at
three sites. Actual inoculum levels ranged from 35-290 CFU/bottle, although there was an instance of a colony count that was
>300 CFU/bottle which was not included in the final data analysis. Bottles were tested with human blood from healthy volunteers and were
held at specified temperatures and times prior to loading into the BacT/ALERT 3D instrument. Percent recovery reflects bottles flagged
positive by the instrument and subculture with colony morphology consistent with the seeded organism.
Table 8. Delayed Entry
Time to Detection from
Incubation Sample Inoculation
Hold Time Inoculum Range
Sample Input Temperature % Recovery
(hours) (Hold Time + Instrument TTD in hours) (CFU/bottle)
(°C)
Mean Range
Control No delay 100.0 (459/459) 14.3 8.5-84.0 35-288
2-8 48 98.6 (292/296) 63.7 57.5-103.2 48-288
Inoculated Test 20-25 24 98.0 (291/297) 31.8 26.2-74.4 50-288
Bottles 20-25 36 91.9 (272/296) 41.8 38.0-70.5 50-290
35-37 8 98.9 (454/459) 16.1 10.2-53.8 35-288
35-37 24 56.6 (259/458)* 28.3 26.0-74.4 35-288
Negative Controls All conditions 0.5 (1/221)† - - -
* CAUTION: Culture bottles held at 35-37°C for 24 hours or longer before loading may not detect microorganisms and should be subcultured.
† One false positive result observed during seeded study. Negative confirmation by Gram stain/subculture.

Within-Laboratory Precision (Repeatability)

Data in Table 9 represent results from in-house seeded studies conducted on 12 days on multiple instruments by multiple operators.
Organisms were grown in the presence of clinically relevant concentrations of antimicrobials to which they are susceptible. In this seeded
study BacT/ALERT PF Plus Culture Bottles were subcultured at least 24 hours after being flagged positive by the instrument. A minimum of
108 replicates were tested for each organism/antimicrobial combination.
Table 9. Within-Laboratory Precision (Repeatability)
Sample Input Range % Recovery Time to Detection (hours)
Organism Antimicrobial (CFU/bottle) Lot 1 Lot 2 Lot 3 Overall Mean Range
C. albicans Fluconazole 140-364 100.0 100.0 100.0 100.0 26.0 22.8-31.3
E. coli Amikacin 26-156 100.0 100.0 100.0 100.0 12.0 11.2-13.0
K. pneumoniae Levofloxacin 108-170 100.0 100.0 100.0 100.0 13.4 11.7-15.2
P. aeruginosa Piperacillin 80-148 100.0 97.2 100.0 99.1 19.2 17.4-24.1
S. pneumoniae Penicillin G 9-505 100.0 100.0 100.0 100.0 13.2 11.6-15.5
S. aureus Vancomycin 94-158 100.0 100.0 100.0 100.0 16.9 14.6-20.3

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Reproducibility
Data in Table 10 represent results from seeded studies conducted at three sites using a target of 162 replicates per site on 3 days with a
minimum of two operators per site. Reproducibility was evaluated on each of nine organisms. Two organisms (C. albicans and
S. pneumoniae) were prepared using serial dilution and the other seven organisms were prepared using BioBall® products. C. albicans and
S. pneumoniae were seeded into the BacT/ALERT PF Plus Culture Bottle at a target inoculum of 100 CFU/bottle, with an acceptable range
of 30-300 CFU/bottle and the other seven organisms at a target range of 1-17 CFU/bottle. The actual inoculum ranged from 6-700 CFU/bottle
for the 30-300 CFU/bottle range, and from 1-270 CFU/bottle for the 1-17 CFU/bottle range. Percent recovery reflects positive flag by the
instrument and Gram-stain/subculture consistent with the seeded organism.
Table 10. Reproducibility
Time to Detection Inoculum
% Recovery
Sample Input (hours) Ranges
Site 1 Site 2 Site 3 Overall Mean Range (CFU/Bottle)
S. aureus 100.0% (18/18) 87.5% (21/24) 100.0% (30/30) 95.8% (69/72) 15.6 14.6-16.7 2-11
C. albicans 100.0% (18/18) 83.3% (30/36) 100.0% (33/33) 93.1% (81/87) 36.6 24.6-76.8 14-700
E. coli 100.0% (27/27) 77.8% (21/27) 100.0% (30/30) 92.9% (78/84) 12.8 11.8-14.1 1-38
P. aeruginosa 100.0% (24/24) 75.0% (18/24) 97.0% (32/33) 91.4% (74/81) 18.4 17.1-21.1 1-11
E. faecalis 100.0% (18/18) 79.2% (19/24) 96.7% (29/30) 91.7% (66/72) 13.9 12.6-15.3 1-15
E. aerogenes 74.4% (29/39) 72.2% (26/36) 85.4% (41/48) 78.1% (96/123) 14.9 11.7-20.8 <1-270*
L. monocytogenes 100.0% (18/18) 100.0% (24/24) 100.0% (30/30) 100.0% (72/72) 24.1 20.4-36.4 1-14
S. enterica 100.0% (24/24) 75.0% (18/24) 100.0% (33/33) 92.6% (75/81) 13.5 2.3-14.8 1-13
S. pneumoniae 100.0% (30/30) 100.0% (36/36) 100.0% (21/21) 100.0% (87/87) 14.2 11.6-18.9 6-500
95.4% (206/216) 83.5% (213/255) 96.9% (279/288) 92.0% (698/759)
Overall 95% CI: 95% CI: 95% CI: 95% CI: -
91.7%, 97.8% 78.4%, 87.9% 94.2%, 98.6% 89.8%, 93.8%
* Plate count of 270 CFU/bottle was arrived at by serial dilution.

These data include repeat testing performed as a result of laboratory errors at a single site (i.e., contaminated subculture). Data excluding the
laboratory errors demonstrated 100% recovery with the exception of E. aerogenes, which exhibited 85% recovery for all sites combined.

BacT/ALERT VIRTUO Microbial Detection Systems

Potentially Interfering Substances
In-house seeded studies were conducted with plasma, blood, and blood clots. Aliquots of each of these fluids also received white blood cells
at concentrations relevant to bacteremia in blood. Testing was conducted with and without microorganisms. These substances neither
interfered with recovery and detection of organisms, nor did they generate false positive results in the absence of organisms.
Analytical Sensitivity: Limit of Detection (LoD)
Data in Table 11 represent results from in-house seeded studies. A minimum of 60 BacT/ALERT PF Plus Bottles were tested per species. At
least 95% detection was achieved at LoD. BacT/ALERT PF Plus Culture Bottles inoculated with H. influenzae received 1 mL of human blood
obtained from a healthy adult population.
Table 11. Analytical Sensitivity: Limit Of Detection (LoD)
BacT/ALERT
Organism Strain ID VIRTUO
(CFU/bottle)
Candida albicans ATCC® 14053™ 7
Enterococcus faecalis NCTC 12697 4
Escherichia coli NCTC 12923 4
Haemophilus influenzae ATCC® 10211™ 3
Pseudomonas aeruginosa ATCC® 9027™ 8
Staphylococcus aureus NCTC 10788 3
Streptococcus pneumoniae ATCC® 6305™ 3

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Delayed Entry
Table 12 presents results from seeded studies using 9 species (Staphylococcus aureus, Candida albicans, Escherichia coli, Acinetobacter
baumannii, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Enterococcus
faecium) at target concentrations of ≤100 CFU/bottle. Actual inoculum levels ranged from 5-84 CFU/bottle. Bottles were tested without and
with 4 mL human blood from healthy volunteers and were held at specified temperatures and times prior to loading into the BacT/ALERT
VIRTUO instrument. Percent recovery reflects bottles flagged positive by the instrument and subculture with colony morphology consistent
with the seeded organism. Negative controls were tested with 10 mL human blood as a more stringent test to evaluate the risk of false
positives.
Table 12. Delayed Entry
Time to Detection from
Incubation Sample Inoculation
Hold Time
Sample Input Temperature % Recovery
(hours) (Hold Time + Instrument TTD in hours)
(°C)
Mean Range
Control No delay 100.0 (259/259) 14.0 8.5-30.0
35-37 8 100.0 (261/261) 16.5 10.9-31.1
Inoculated Test 32.1
20-25 24 100.0 (261/261) 27.6-46.6
Bottles
20-25 36 99.6 (260/261) 42.0 37.7-65.1
2-8 48 96.5 (247/256)* 61.3 57.2-77.9
Negative Controls All conditions 0.0 (0/64)† - -
* 8/9 Negative bottles were observed when tested with N. meningitides.
† Negative controls tested with 10 mL human blood.
CAUTION: Culture bottles held at room temperature for longer than 24 hours before loading may not detect microorganisms and should be
subcultured.

Within-Laboratory Precision (Repeatability)

Data in Table 13 represent results from in-house seeded studies conducted for 20 days using multiple instruments and tested by multiple
operators. A minimum of 60 replicates were tested for each organism and lot tested.
Table 13. Within-Laboratory Precision (Repeatability)
Range % Recovery Time to Detection (hours)
Organism
(CFU/bottle) Lot 1 Lot 2 Lot 3 Overall Mean Range
100.0 100.0 100.0 100.0
Candida albicans 3-30 27.1 21.4-40.0
(141/141) (144/144) (60/60) (345/345)
100.0 100.0 100.0 100.0
Enterococcus faecalis 3-16 10.9 9.6-12.4
(162/162) (162/162) (60/60) (384/384)
100.0 100.0 100.0 100.0
Escherichia coli 8-20 9.6 8.7-10.8
(153/153) (153/153) (60/60) (366/366)
100.0 100.0 100.0 100.0
Haemophilus influenzae* 1-24 13.6 11.0-21.6
(162/162) (162/162) (60/60) (384/384)
100.0 100.0 100.0 100.0
Pseudomonas aeruginosa 5-17 14.7 12.8-17.6
(162/162) (162/162) (60/60) (384/384)
100.0 100.0 100.0 100.0
Staphylococcus aureus 5-16 12.7 11.5-15.0
(171/171) (171/171) (60/60) (402/402)
100.0 100.0 100.0 100.0
Streptococcus pneumoniae 1-29 12.0 9.6-15.6
(162/162) (162/162) (60/60) (384/384)
* Tested with 4 mL blood.

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BacT/ALERT 3D and BacT/ALERT VIRTUO Systems Comparative Data

Analytical Sensitivity: Growth Performance
Data in Table 14 represent results from in-house seeded studies with blood (4 mL) obtained from healthy human donors. A single strain was
tested for each species at target inoculum levels of ≤30 CFU/bottle. The actual inoculum levels ranged from 7-30 CFU/bottle for both
BacT/ALERT 3D and BacT/ALERT VIRTUO. Two positive bottles were subcultured per organism to assess purity. The species listed are
representatives of clinically prevalent organisms in blood cultures.
Table 14. Analytical Sensitivity: Growth Performance
BacT/ALERT PF Plus BacT/ALERT VIRTUO - BacT/ALERT PF Plus BacT/ALERT 3D -
4 mL Blood 4 mL Blood
% Recovery Average Time to Detection % Recovery Average Time to Detection
Organism (n) CFU/Bottle Mean Range (n) CFU/Bottle Mean Range
100.0 100.0
Abiotrophia defectiva 23 14.8 14.2-16.1 15 23.1 19.0-28.6
(9/9) (4/4)
Aggregatibacter 100.0 100.0
21 29.8 27.2-32.2 21 32.6 28.1-34.8
actinomycetemcomitans (9/9) (9/9)
100.0 100.0
Campylobacter jejuni 7 38.2 34.2-41.5 7 40.6 37.7-43.2
(9/9) (9/9)
100.0 100.0
Candida albicans 11 26.9 23.4-29.8 11 28.3 26.6-31.0
(9/9) (9/9)
100.0 100.0
Candida glabrata 7 41.7 38.4-44.5 7 49.4 45.6-55.9
(9/9) (9/9)
100.0 100.0
Candida krusei 20 16.8 16.1-17.8 20 19.1 18.7-19.7
(9/9) (9/9)
100.0 100.0
Cardiobacterium hominis 12 41.6 39.7-44.5 12 52.9 50.9-55.0
(9/9) (9/9)
100.0 100.0
Corynebacterium jeikeium 8 39.5 29.9-50.7 8 84.4 52.1-112.8
(9/9) (9/9)
100.0 100.0
Cryptococcus neoformans 30 56.6 49.5-65.8 30 57.8 56.4-59.0
(7/7) (9/9)
100.0 100.0
Eikenella corrodens 24 21.2 20.1-22.5 24 24.7 24.0-25.4
(9/9) (9/9)
100.0 100.0
Enterobacter aerogenes 10 10.7 10.3-11.1 10 12.4 12.0-12.7
(9/9) (9/9)
100.0 100.0
Enterococcus faecalis 11 10.1 9.4-10.8 11 12.2 12.0-12.7
(9/9) (9/9)
100.0 100.0
Escherichia coli 9 9.0 8.6-9.6 9 10.9 9.1-11.3
(9/9) (9/9)
100.0 100.0
Haemophilus influenzae 18 14.8 13.5-16.3 18 17.8 17.0-18.5
(9/9) (9/9)
100.0 100.0
Klebsiella pneumoniae 9 9.3 8.8-9.9 9 11.8 11.8-12.0
(9/9) (9/9)
100.0 100.0
Listeria monocytogenes 19 18.9 18.2-20.0 19 20.5 20.2-20.9
(9/9) (9/9)
100.0 100.0
Micrococcus luteus 24 31.2 30.0-32.4 24 33.2 32.4-33.8
(9/9) (9/9)
100.0 100.0
Neisseria meningitidis 8 22.2 21.1-23.8 8 23.8 22.1-25.0
(9/9) (9/9)
100.0 100.0
Proteus vulgaris 22 11.4 11.0-12.1 22 13.4 13.2-13.4
(9/9) (9/9)
100.0 100.0
Pseudomonas aeruginosa 12 14.1 13.8-14.6 12 16.8 16.6-17.0
(9/9) (9/9)
100.0 100.0
Salmonella enterica 9 11.0 10.6-11.4 9 13.1 12.5-13.9
(9/9) (9/9)
100.0 100.0
Serratia marcescens 10 11.2 10.7-12.2 10 13.0 12.5-13.2
(9/9) (9/9)
100.0 100.0
Staphylococcus aureus 14 10.4 10.1-10.8 15 13.3 12.7-13.9
(9/9) (9/9)
100.0 100.0
Staphylococcus epidermidis 11 14.6 14.1-15.8 11 17.2 16.6-17.8
(9/9) (9/9)
100.0 100.0
Stenotrophomonas maltophilia 22 65.1 24.8-83.9 22 33.5 31.4-37.0
(7/7) (9/9)
100.0 100.0
Streptococcus agalactiae 13 11.0 10.5-12.2 13 14.3 13.9-14.6
(9/9) (9/9)
100.0 100.0
Streptococcus mitis 17 8.8 8.6-9.0 17 11.9 11.5-12.0
(9/9) (9/9)
100.0 100.0
Streptococcus pneumoniae 22 11.2 10.6-12.1 22 13.8 13.4-14.2
(9/9) (9/9)
100.0 100.0
Streptococcus pyogenes 16 10.4 9.8-10.9 16 12.7 12.5-13.0
(9/9) (9/9)

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Clinical Study Results (Blood Cultures)

Results compare BacT/ALERT VIRTUO to BacT/ALERT 3D with BacT/ALERT PF Plus bottles for blood cultures (for all compliant pairs).
A multi-center clinical study was conducted at three different geographic sites in the U.S. comparing the performance of the BacT/ALERT
VIRTUO and BacT/ALERT 3D for aerobic culture pairs in which each bottle was filled with up to 4 mL of blood and [1) if the total volume of
blood collected was > 5 mL, the blood volume of the bottle with the smallest volume was within 20% of that of the bottle with the largest
volume or 2) if the total volume of blood collected was <5 mL, the blood volume of the bottle with the smallest volume was within 50% of that
of the bottle with the largest volume] (compliant pairs). A total of 161 bottle pairs were obtained from 62 pediatric patients suspected of blood
stream bacterial/yeast infections. Subcultures of both bottles were performed when either bottle in the set was determined to be positive by the
BacT/ALERT VIRTUO or BacT/ALERT 3D System. A pair of bottles was determined to have a positive status if the subculture of either the
BacT/ALERT VIRTUO or BacT/ALERT 3D PF Plus Culture Bottle was positive. A culture bottle was determined to be a “True Positive” if the
culture was flagged positive by the BacT/ALERT VIRTUO or BacT/ALERT 3D System and resulted in growth of the isolate upon subculture of
this bottle. True positive rates were calculated for the BacT/ALERT VIRTUO PF Plus and BacT/ALERT 3D PF Plus Culture Bottles, and the
ratio of BacT/ALERT VIRTUO PF Plus true positives to BacT/ALERT 3D PF Plus true positives was calculated to compare performance.
Clinical isolates recovered were classified as significant, contaminant, or unknown based on determination by the clinical trial sites.
A total of 2 isolates were recovered from all compliant aerobic blood culture pairs with a positive status. There were a total of 2 bottle pairs that
recovered a single isolate by subculture of BacT/ALERT VIRTUO or BacT/ALERT 3D PF Plus Culture Bottles. The total population reported in
Table 15 comprises the 2 isolates recovered from positive bottle pairs and 159 negative bottle pairs for a total of 161 results. The BacT/ALERT
VIRTUO PF Plus Culture Bottle detected a total of 1 isolate compared to the BacT/ALERT 3D PF Plus Culture Bottle that detected 1 isolate.
Of the significant isolates, the BacT/ALERT VIRTUO PF Plus Culture Bottle detected a total of 1 isolate compared to the BacT/ALERT 3D PF
Plus Culture Bottle that detected 1 isolate. One false positive was identified by subculture of positive BacT/ALERT VIRTUO PF Plus Culture
Bottles in the study population 0.62% (1/161). No false positives were identified by subculture of positive BacT/ALERT 3D PF Plus Culture
Bottles in the study population (0/161).
The following table compares results of the BacT/ALERT VIRTUO to BacT/ALERT 3D blood cultures for all compliant BacT/ALERT PF Plus
blood culture bottles that yielded a single isolate on subculture (Table 15). No compliant aerobic blood culture pairs with a positive status
yielded multiple isolates on subculture.
Table 15. Compliant – Single Isolates
% of BacT/ALERT
BacT/ALERT BacT/ALERT % of BacT/ALERT Ratio of
VIRTUO True 95% CI
Clinical Determination VIRTUO True 3D True 3D True Positives True
Positives in (LCL, UCL)
Positives Positives in Population Positives
Population
Significant 1 0.6 (1/161) 1 0.6 (1/161) 1.000 -1.772, 3.772*
Contaminant 0 0.0 (0/161) 0 0.0 (0/161) - -
Unknown 0 0.0 (0/161) 0 0.0 (0/161) - -
Total 1 0.6 (1/161) 1 0.6 (1/161) 1.000 -1.772, 3.772*
* Since the confidence interval contains a negative value and the ratio cannot be negative, the interval does not provide a meaningful interpretation.

A comparative yield of microorganisms (number of isolates) from BacT/ALERT VIRTUO and BacT/ALERT 3D recovered on subculture of
BacT/ALERT PF Plus culture bottles is presented in Table 16.
Table 16. Comparative Yield Of Microorganisms From BacT/ALERT VIRTUO And BacT/ALERT 3D (Number of Isolates) – Blood
Cultures
Group BacT/ALERT VIRTUO BacT/ALERT 3D
Enterobacteriaceae 1 0
Enterococcus spp. - -
Yeasts - -
Non-fermentative Gram-Negative Bacilli - -
Other Gram-Positive - -
Coagulase-Negative Staphylococcus - -
Staphylococcus aureus 0 1
Streptococcus spp. - -
Other - -

In this clinical study, there were 196 pairs of BacT/ALERT VIRTUO and BacT/ALERT 3D Culture Bottles with negative instrument results for
both systems after 5 days of incubation. Among these pairs, terminal subcultures were performed for 50 pairs, and no false negative result by
either BacT/ALERT VIRTUO or BacT/ALERT 3D was observed; subculture on BacT/ALERT 3D bottles alone was performed for 1 pair, and no
false negative result was observed; both subcultures were not performed for 145 pairs of bottles. A culture bottle was determined to be false
negative if the bottle result was negative by the instrument and resulted in growth upon subculture of the bottle.
Table 17. Summary Of Percent False Negatives From Aerobic Blood Culture Pairs That Were Flagged Negative By Both Instruments
Subculture Performed Subculture Performed % False Negative % False Negative
BacT/ALERT VIRTUO BacT/ALERT 3D BacT/ALERT VIRTUO BacT/ALERT 3D
Yes Yes 0.0 (0/50) 0.0 (0/50)
No Yes - 0.0 (0/1)

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Clinical Study Results (Blood Cultures) – Low Fill BacT/ALERT FA Plus Culture Bottles
Results compare BacT/ALERT VIRTUO to BacT/ALERT 3D with low fill BacT/ALERT FA Plus bottles for blood cultures (for all compliant
pairs).
Due to the low numbers of positive BacT/ALERT PF Plus bottles observed in the clinical study, low fill volume BacT/ALERT FA Plus bottles
were evaluated as a surrogate for BacT/ALERT PF Plus as both are equal in terms of the media composition. Note that the bottle labeling for
BacT/ALERT PF Plus and BacT/ALERT FA Plus bottles provide the recommended blood volumes for each bottle type and differentiate their
respective use in a clinical setting. A multi-center clinical study was conducted at three different geographic sites in the U.S. and Canada
comparing the performance of the BacT/ALERT VIRTUO and BacT/ALERT 3D for aerobic culture pairs in which each bottle was filled with up
to 4 mL of blood and in which the blood volume of the bottle with the smallest volume was within 30% of that of the bottle with the largest
volume (compliant pairs). A total of 379 bottle pairs were obtained from 292 adult patients suspected of blood stream bacterial/yeast
infections. Subcultures of both bottles were performed when either bottle in the set was determined to be positive by the BacT/ALERT
VIRTUO or BacT/ALERT 3D System. A pair of bottles was determined to have a positive status if the subculture of either the BacT/ALERT
VIRTUO or BacT/ALERT 3D FA Plus Culture Bottle was positive. A culture bottle was determined to be a “True Positive” if the culture was
flagged positive by the BacT/ALERT VIRTUO or BacT/ALERT 3D System and resulted in growth of the isolate upon subculture of this bottle.
True positive rates were calculated for the BacT/ALERT VIRTUO FA Plus and BacT/ALERT 3D FA Plus Culture Bottles, and the ratio of
BacT/ALERT VIRTUO FA Plus true positives to BacT/ALERT 3D FA Plus true positives was calculated to compare performance. Clinical
isolates recovered were classified as significant, contaminant, or unknown based on determination by the clinical trial sites.
A total of 40 isolates were recovered from all compliant aerobic blood culture pairs with a positive status. There were a total of 40 bottle pairs
that recovered a single isolate by subculture of BacT/ALERT VIRTUO or BacT/ALERT 3D FA Plus Culture Bottles. The total population
reported in Table 18 comprises the 40 isolates recovered from positive bottle pairs and 339 negative bottle pairs for a total of 379 results. The
BacT/ALERT VIRTUO FA Plus Culture Bottle detected a total of 33 isolates compared to the BacT/ALERT 3D FA Plus Culture Bottle that
detected 32 isolates. Of the significant isolates, the BacT/ALERT VIRTUO FA Plus Culture Bottle detected a total of 28 isolates compared to
the BacT/ALERT 3D FA Plus Culture Bottle that detected 29 isolates. No false positives were identified by subculture of positive BacT/ALERT
VIRTUO FA Plus Culture Bottles in the study population (0/379). No false positives were identified by subculture of positive BacT/ALERT 3D
FA Plus Culture Bottles in the study population (0/379).
The following table compares results of the BacT/ALERT VIRTUO to BacT/ALERT 3D blood cultures for all compliant low fill BacT/ALERT FA
Plus blood culture bottles that yielded a single isolate on subculture (Table18). No compliant aerobic blood culture pairs with a positive status
yielded multiple isolates on subculture.
Table 18. Low Fill BacT/ALERT FA Plus – Blood – Compliant – Single Isolates
% of BacT/ALERT
BacT/ALERT BacT/ALERT % of BacT/ALERT Ratio of
VIRTUO True 95% CI
Clinical Determination VIRTUO True 3D True 3D True Positives True
Positives in (LCL, UCL)
Positives Positives in Population Positives
Population
Significant 28 7.4 (28/379) 29 7.7 (29/379) 0.966 0.790, 1.142
Contaminant 4 1.1 (4/379) 3 0.8 (3/379) 1.333 -
Unknown 1 0.3 (1/379) 0 0.0 (0/379) - -
Total 33 8.7 (33/379) 32 8.4 (32/379) 1.031 0.790, 1.272

A comparative yield of microorganisms (number of isolates) from BacT/ALERT VIRTUO and BacT/ALERT 3D recovered on subculture of
compliant low fill BacT/ALERT FA Plus culture bottles is presented in Table 19.
Table 19. Comparative Yield Of Microorganisms (Number of Isolates) – Low Fill BacT/ALERT FA Plus Blood Cultures
Group BacT/ALERT VIRTUO BacT/ALERT 3D
Enterobacteriaceae 8 9
Enterococcus spp. 3 2
Yeasts 2 1
Non-fermentative Gram-Negative Bacilli 2 1
Other Gram-Positive 0 1
Coagulase-Negative Staphylococcus 5 5
Staphylococcus aureus 6 7
Streptococcus spp. 6 5
Other 1 1

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BacT/ALERT® PF Plus 9313400 E – en – 2017-04

In this clinical study, there were 492 pairs of BacT/ALERT VIRTUO and BacT/ALERT 3D Culture Bottles with negative instrument results for
both systems after 5 days of incubation. Among these pairs, terminal subcultures were performed for 242 pairs, and no false negative result by
either BacT/ALERT VIRTUO or BacT/ALERT 3D was observed; subculture on BacT/ALERT VIRTUO bottles alone was performed for 4 pairs,
and no false negative result was observed; subculture on BacT/ALERT 3D bottles alone was performed for 3 pairs, and no false negative
result was observed; both subcultures were not performed for 243 pairs of bottles. A culture bottle was determined to be false negative if the
bottle result was negative by the instrument and resulted in growth upon subculture of the bottle.
Table 20. Summary Of Percent False Negatives From Low Fill BacT/ALERT FA Plus Aerobic Blood Culture Pairs That Were Flagged
Negative By Both Instruments
Subculture Performed Subculture Performed % False Negative % False Negative
BacT/ALERT VIRTUO BacT/ALERT 3D BacT/ALERT VIRTUO BacT/ALERT 3D
Yes Yes 0.0 (0/242) 0.0 (0/242)
Yes No 0.0 (0/4) -
No Yes - 0.0 (0/3)

Summary of False Positive Results

A culture bottle was determined to be a false positive if the culture was flagged positive by the BacT/ALERT VIRTUO or BacT/ALERT 3D
System and was negative upon subculture of the bottle. The study population consisted of culture pairs that received blood volumes of ≤4 mL
(BacT/ALERT PF Plus and low fill BacT/ALERT FA Plus bottles). The false positive results were identified by subculture of positive
BacT/ALERT VIRTUO bottles and positive BacT/ALERT 3D bottles, comprising proportions of the study populations based on the total
numbers of corresponding blood cultures.
Table 21. Summary Of False Positive Results
% False Positive % False Positive
Bottle Type – Specimen Type
BacT/ALERT VIRTUO BacT/ALERT 3D
BacT/ALERT PF Plus - Blood 0.50 (1/202) 0.00 (0/202)
Low Fill BacT/ALERT FA Plus – Blood 0.00 (0/550) 0.00 (0/550)

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BacT/ALERT® PF Plus 9313400 E – en – 2017-04

REFERENCES INDEX OF SYMBOLS

1. Thorpe TC, Wilson ML, Turner JE, DiGuiseppi JL, Willert M, Mirrett S, et Symbol Meaning
al: BacT/ALERT: an Automated Colorimetric Microbial Detection
Catalogue number
System. J Clin Micro 1990;28(7), 1608-1612.
2. Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Legal Manufacturer
Edition. U.S. Department of Health and Human Services Centers for
Disease Control and Prevention and National Institutes of Health. Fifth Date of Manufacture
Edition. US Government Printing Office. Washington: Feb 2007.
3. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC.
Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. 2006, pp. Temperature limit
446,590.
4. Baron EJ, Weinstein MP, Dunne Jr. WM, Yagupsky P, Welch DF,
Wilson DM. 2005. Cumitech 1C, Blood Cultures lV. Coordinating ed. Use by date
Baron EJ. ASM Press, Washington, DC.
5. CLSI. Principles and Procedures for Blood Cultures; Approved Batch code
Guideline. CLSI document M47-A. Wayne, PA: Clinical and Laboratory
Standards Institute; 2007. Consult Instructions for Use
6. CLSI. Quality Control for Commercially Prepared Microbiological Culture
Media; Approved Standard—Third Edition. CLSI/NCCLS document Contains sufficient for <n> tests
M22-A3. Wayne, PA: NCCLS; 2004.
7. Kondratovich M. Comparing Two Medical Tests When Results of Authorized representative in the European
Reference Standard Are Unavailable for Those Negative via Both Tests. Community
J. Biopharm Stat 2008;18:1,145-166.
This way up
LIMITED WARRANTY
In Vitro Diagnostic Medical Device
bioMérieux warrants the performance of the product for its stated
intended use provided that all procedures for usage, storage and Do not reuse
handling, shelf life (when applicable), and precautions are strictly
followed as detailed in the instructions for use (IFU).
Latex-free
Except as expressly set forth above, bioMérieux hereby disclaims
all warranties, including any implied warranties of merchantability For US Only: Caution: US Federal Law restricts this
device to sale by or on the order of a licensed
and fitness for a particular purpose or use, and disclaims all liability,
practitioner.
whether direct, indirect or consequential, for any use of the reagent,
software, instrument and disposables (the “System”) other than as
set forth in the IFU. Instructions for use provided in the kit or downloadable from
www.biomerieux.com/techlib
AVAILABILITY
bioMérieux
BacT/ALERT® PF Plus
100/case 410853
For technical assistance in the USA, contact bioMérieux Customer
Service at 1-800-682-2666. Outside the USA, contact your local
bioMérieux representative.

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BacT/ALERT® PF Plus 9313400 E – en – 2017-04

REVISION TABLE
This section contains a summary of changes made to the BacT/ALERT PF Plus Instructions for Use.
Revision Date Revision Number Change Type Change Summary
2017-04 9313400 E Content Change: Addition of VIRTUO information throughout, including Expected Values, Neutralization
of Antimicrobials and Performance Characteristics of the Test (Tables 11-21)
sections.
2016-04 9309505 D Content Change: Reagents
- Update to composition information
- Clarification of expiration date
Specimen Collection and Preparation - Addition of Caution regarding bottle pressure
Quality Control - Addition of Caution regarding LIS and bottle type abbreviations
Addition of Rx-only caution and symbol for US customers
Administrative: Limited Warranty - Addition of statement
Index of Symbols – Update to reflect new symbols on product
2013-04 9305130 C Content Change: Intended Use, Specimen Collection and Preparation, Expected Values,
Neutralization of Antimicrobials, Performance Characteristics of the Test - Revised
text to include additional information on product performance based on clinical studies
Limitations of the Test - Added Limitations 2, 8, and 9 following FDA review
Administrative: Reagents - Moved Cautions to Limitations of the Test section
NOTE: Minor typographical, grammar, and formatting changes are not included in the revision history.
Change Type categories:
- Correction = Correction of documentation anomalies.
- Content Change = Implementation of new and modified (updated) intended use and performance characteristics.
- Administrative = Implementation of non-technical changes noticeable to the user.

BIOMÉRIEUX, the BIOMÉRIEUX logo, BacT/ALERT, BioBall, FAN, and VIRTUO are used, pending, and/or registered trademarks belonging to bioMérieux, or
one of its subsidiaries, or one of its companies.
This product may be protected by one or more patents, see: http://www.biomerieux-usa.com/patents.
The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection.
CLSI is a registered trademark belonging to Clinical and Laboratory Standards Institute.
Any other name or trademark is the property of its respective owner.
©BIOMÉRIEUX 2011, 2013, 2016, 2017

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