BOSCH 148838 Vivalytic Noro Rotavirus C. Diff Test Instruction Manual
- August 11, 2024
- Bosch
Table of Contents
BOSCH 148838 Vivalytic Noro Rotavirus C. Diff Test Instruction Manual
Introduction
Clostridioides difficile (C. difficile) is a gram-positive, spore-forming anaerobic bacillus consisting of toxigenic and non-toxigenic strains. C. difficile is one of the most common pathogens of nosocomial diarrhea, especially in patients with antibiotic-associated diarrhea. In recent years, the number and severity of cases has increased in Europe and worldwide.1 The symptoms of C. difficile infection (CDI) ranges from mild diarrhea to severe life-threatening pseudomembranous colitis, although colonization of C. difficile does not necessarily lead to a symptomatic infection. The disruption of the balance of gut microorganisms e.g. due to antibiotically treatment may cause toxigenic C. difficile to establish, dominate colonization and might be the start of CDI.2
Factors involved in the pathogenesis of C. difficile, are the genes tcdA and tcdB that code for toxin A (enterotoxin) and toxin B (cytotoxin) which are located in a 19.6 kb chromosomal region called the pathogenicity locus (PaLoc). Most pathogenic strains are toxin A and B positive (A+B+), but some pathogenic variant isolates may be toxin A negative and B positive (A-B+). They are the cause of C. difficile-associated diseases.3 Some strains of toxigenic C. difficile also produce a toxin called C. difficile transferase (CDT), or binary toxin.
Norovirus belongs to the virus class Caliciviridae and is known to be a seasonal disease. It is a highly contagious pathogen that is responsible for the majority of cases of acute gastroenteritis worldwide. Norovirus infections in humans are mainly caused by genogroup II and I, while genogroup II being the predominant cause of Norovirus outbreaks. Infections of the gastrointestinal tract by Norovirus are characterized by sudden onset symptoms such as nausea, vomiting, diarrhea, and abdominal pain. The virus is particularly prevalent in communal settings such as hospitals, schools, and nursing homes, where it can easily spread from person to person. The illness typically lasts only a few days but can cause more severe complications in immunocompromised individuals or the elderly.5,6,7
Rotavirus belongs to the virus class Reoviridae. It is a significant cause of acute gastroenteritis, particularly in infants and young children. Its viral infection of the gastrointestinal tract is characterized by symptoms such as severe diarrhea, vomiting, fever, and dehydration. Rotavirus-associated gastroenteritis can lead to severe complications, including hospitalization and even death, making it a major public health concern. Efforts to prevent and control rotavirus infections, such as through vaccination programs, have been crucial in reducing the burden of this disease. Acute gastroenteritis caused by both Norovirus and Rotavirus are self-limiting diseases although if untreated can cause a critical course of disease especially in the youngest or elderly. 7,8,9,10
Package Contents
15 Vivalytic Noro-, Rotavirus, C.diff test cartridges for the detection of Clostridioides difficile (tcdA/tcdB toxin genes), human Norovirus genogroup I/ II and Rotavirus type A.
Pathogen List
Clostridioides difficile
(tcdA/tcdB)
Norovirus genogroup I/II
Rotavirus Type A
Intended Use
The Vivalytic Noro-, Rotavirus, C.diff test is an automated qualitative in vitro diagnostic test based on real-time polymerase chain reaction (PCR) for the detection of nucleic acids from toxigenic Clostridioides difficile (toxin genes tcdA/tcdB), Human Norovirus genogroup I/ II and Human Rotavirus type A from swabs samples from liquid or soft human stool to aid in the diagnosis of acute gastrointestinal infections of symptomatic individuals.
Results should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive results do not exclude co-infection with other pathogens. The agent(s) detected may not be the definite cause of disease. Negative results do not exclude a C. difficile, Norovirus, Rotavirus infection or another gastrointestinal infection. Results must be clinically correlated with patient history, clinical observations and epidemiological information. Other diagnostic information is necessary to determine patient infection status. Intended for use with a Vivalytic one analyser by healthcare professionals only in laboratory settings.
Safety Information
These Instructions for Use contain test-specific information only. For additional warnings and instructions refer to the Instructions for Use provided with your Vivalytic one analyser (chapter device safety information). Only use Vivalytic cartridges and accessories approved for the Vivalytic one analyser. Take care to avoid any contamination when handling patient samples and cartridges. When sample was spilled on the cartridge, do not use the cartridge, and dispose it.
For in vitro diagnostic use by trained healthcare professional.
**WARNING**
- Always follow good laboratory practice to ensure the proper performance of this test.
- Make sure to wear appropriate personal protective equipment (PPE).
- Do not use a cartridge if the sealed pouch or the cartridge itself is visibly damaged.
- Do not touch or scratch the detection area of the cartridge.
- Do not reuse a cartridge.
- Do not use expired cartridges. The expiration date can be found on the packaging and the cartridge label
- Do not wait longer than 1 5 minutes after opening the cartridge pouch to begin the test.
- This maintains hygiene and avoids performance loss due to humidity. Prolonged exposure to humidity has a negative impact on test performance.
- Do not shake a cartridge that contains a sample.
- Do not turn the cartridge upside down.
- Place the cartridge on a clean and flat surface only.
- Do not use sample types, media and volumes that are not approved for the test.
- Always follow good laboratory practice to ensure the proper performance of this test.
- Biological specimens, transfer devices, and used cartridges should be considered capable of transmitting infectious agents requiring standard precautions. Handle potentially infectious patient samples and cartridges according to national laboratory standards and dispose samples and cartridges according to regional and laboratory standards.
- Be compliant with the national safety regulations and practices.
Note: Further information can be found in the safety data sheet (SDS) of the product. Please contact the customer support of your local distributor.
Additional Equipment & Consumables Required but not Provided • Bosch Vivalytic one analyser (reference number F09G300115)
-
Pipettor (100–1000 μl)
-
Sterile filter pipette tips 100 –1000 µl
-
Swab collection kits
– Regular Flocked Swab FLOQSwabs® 552C (COPAN Italia s.p.a.) -
Transport medium
– eNAT® 606C (COPAN Italia s.p.a.) -
Suitable protective clothing
Test Principle
Vivalytic Noro-, Rotavirus, C. diff is a qualitative real-time PCR based test.
Storage und Usage Conditions
Product is stable until the expiry date if stored at +15 °C to +25 °C. Storage
and usage conditions can be taken either from the cartridge, pouch, or box
label. Cartridge has to be used at +15 °C to +25 °C, relative humidity <65 %,
within 15 min upon pouch opening. This maintains hygiene and avoids
performance loss due to humidity. Prolonged exposure to humidity has a
negative impact on test performance.
Reagents
All reagents necessary for the sample processing are integrated into the
cartridge. The processing includes cell lysis, nucleic acid extraction, DNA
amplification and detection.
Reagents are PCR bead, binding buffer, washing buffer, and elution buffer. The
PCR bead contains the DNA polymerase, primers, and probes. Binding buffer
facilitates binding of nucleic acids during the purification process. Washing
buffer is a formulation of different salts and solvents to remove impurities
e. g. proteins during the extraction process. Elution Buffer is a low-salt
buffer and contains the purified nucleic acids at the end of the extraction
process.
Sample Type/Medium
The test is intended for use with stool swab samples in eNAT® medium (Regular
Flocked Swab FLOQSwabs® 552C, eNAT® transport medium Ref. 606C, COPAN Italia
s.p.a.).
Collect and store samples as indicated in the manufacturer’s instructions.
In case the sample is not processed immediately after sample collection,
nucleic acids will be preserved in eNAT® transport medium for up to 4 weeks at
room temperature and at 4°C and up to 6 months at -20° C to -80° C (see
Instructions for Use eNAT® transport medium11).
Sample Preparation
Use a Regular Flocked Swab FLOQSwabs® (COPAN Italia s.p.a.) to collect a small
amount of stool by inserting the tip of the flocked swab into the stool sample
and rotate it. Bloody, slimy, or watery area of stools should be selected and
collected. After collection, examine the swab to make sure there is fecal
material visible on the tip. In case it is not, again insert the flocked swab
into the stool sample and rotate taking care that all the area of the swab tip
is in contact with the sample. Make sure that the swab is just covered with
stool and remove excess stool by gently rotating the swab against the rim of
the sample. After collection transfer the swab into the 2 ml Copan eNAT® tube
with eNAT® transport medium. Hold the swab shaft between thumb and finger,
mash and mix the stool specimen against the side of the tube to evenly
disperse and suspend the specimen in the preservation medium. Hold the swab
shaft close to the rim of the tube, bend it at a 180 degrees angle to break it
off at the marked breakpoint. Discard the broken upper part of the swab shaft
and tighten the cap. Shake the sample tube containing the swab sample and
eNAT® medium (COPAN Italia s.p.a.) thoroughly at least for 10 seconds for
homogenization. Use a pipettor in order to fill 300 μl of homogenized patient
sample in the sample input of the cartridge. Ensure to only pipet from the
supernatant (top of the sample) to prevent the carryover of stool particles.
In case of an excess amount of particles in the sample it is recommended to
place the sample tube on a flat surface and let the particles sediment for 5
minutes.
Do not use viscous samples that are difficult to pipette.
Test Result
After automatic processing of the sample with the Vivalytic one analyser the
test result is shown on the screen of the Vivalytic one analyser. The time to
result is about 58 min. For high titer specimens results are available after
less than 41 min and the test run can be terminated earlier (see Chapter Test
Termination).
The sample is classified either as C. difficile positive, Rotavirus and C.
difficile positive, Norovirus and C. difficile positive, Norovirus and
Rotavirus and C. difficile positive, Rotavirus positive, Norovirus and
Rotavirus positive, Norovirus positive, negative or invalid. In case of a
positive detection of a pathogen, the test is considered valid even if the
Human Control is negative.
Detection of the human cell based whole process control (Human Control) in negative samples shows a successful extraction procedure and excludes an inhibition of the PCR reaction. Interpretation of results is shown in the table below.
1 Retesting is recommended
PCR – Curve and Cq Value
Real-time PCR curves (software-modified) are shown and classified as positive
or negative by the software. In case of positive curves, the respective Cq
value is displayed. Inconclusive results are marked by the software ( ).
Retesting is advised.
Invalid or Failed Tests
A test is rated as invalid if neither target DNA nor Human Control is
detected. Possible reasons for an invalid run might be poor sample quality due
to a partial or complete absence of human cellular material in the sample.
Results are displayed for an invalid test but are not allowed to be used for
diagnostic interpretation. Pay attention to use the correct sample type,
sample collection and storage of the sample and cartridges prior to the test
run. If required, repeat the analysis with a new sample. In case of a failed
test, first check for correct operating conditions of the Vivalytic one
analyser (refer to Vivalytic one analyser’s Instructions for Use). Restart the
Vivalytic one analyser. If the problem persists, contact the customer support
of your local distributor.
Test Termination
As soon as a valid, positive result is shown for one of the three pathogens on
the screen, the user has the option to finish the test.
Test Report
In the printed test report, pathogen, results, control and information on
user, patient and Vivalytic one analyser are listed with a signature field.
Notice to Users in EU
Any serious incident that has occurred in relation to the device, should be
reported to the manufacturer and the competent authority of the Member State
in which the user and/or patient is established.
Limitations
The results of the Vivalytic Noro-, Rotavirus, C.diff test must be interpreted
by a trained healthcare professional only. The results of the Vivalytic Noro-,
Rotavirus, C.diff test must not be used as the sole parameter for diagnosis.
- A negative result does not exclude pathogens being present in the sample at a level below assay sensitivity or other pathogens being present not covered by this assay.
- There is a risk of false negative or false positive results due to improperly collected, transported, or handled samples.
- In borderline cases atypical PCR characteristics (e. g. flat curve with low or high Cq-value) can occur. In case of atypical characteristics results are not allowed to be used for diagnostic interpretation. Inconclusive results are marked by the software. Retesting is advised.
- Vivalytic Noro-, Rotavirus, C.diff is a qualitative real-time PCR test and does not provide a quantitative result.
- A positive result does not necessarily mean that viable pathogens are present.
- The test detects C. difficile carrying tcdA/tcdB genes, but does not differentiate between toxin producing and non-toxin producing strains
- A negative result does not preclude a Norovirus, Rotavirus, C. difficile or co-infection. It is recommended that negative tested samples are interpreted in the context with additional laboratory data
- An excess amount of stool may have inhibitory effects on the assay performance.
- A reduced performance was observed for tcdA+/tcdB- C. difficile strains
- An excess amount of Rotavirus A virus particles can lead to a reduced performance of the C. difficile PCR
- No exclusivity for Rotavirus Type C (porcine Rotavirus) could be achieved. Therefore a misidentification of Rotavirus Type C as Type A cannot be excluded.
Analytical Performance Evaluation
Analytical Sensitivity (Limit of Detection)
The limit of detection of the Vivalytic Noro-, Rotavirus, C. diff test was
determined as the lowest concentration of analyte that can be consistently
detected (≥95% of samples tested under routine laboratory conditions using a
defined type of sample). (Table 1)
Inclusivity
To evaluate inclusivity, an in silico analysis (BLAST alignment) of the
genomic sequence of various relevant Noroviruses, Rotaviruses and
Clostridioides difficile strains against the sequence of the PCR primers and
hydrolysis probe used in the Vivalytic Noro-, Rotavirus, C. diff test for
amplification and detection of the respective pathogens was performed.
Inclusivity could be shown for strains listed in Table 2.
Exclusivity/ Analytical Specificity
To exclude cross-reactivity (exclusivity), an in silico analysis (BLAST
alignment) of the target region of Norovirus, Rotavirus and Clostridioides
difficile against the genomic sequence of various other pathogens representing
common gastrointestinal pathogens or closely related species was conducted.
For the detection system of Rotavirus, sequence matches in the probe and
primer area could be detected for porcine Rotavirus (group C), concluding a
possible amplification. There was no evidence of an interference for the
detection system of Norovirus and Clostridioides difficile. (Table 3).
Reproducibility
The reproducibility of the Vivalytic Noro-, Rotavirus, C. diff test was
established using a panel with 3 different concentrations of Norovirus,
Rotavirus and Clostridioides difficile. At 3 test sites, each mix was tested
on the same set of Vivalytic instruments by the same operator with 3 LOTs in 4
replicates on 3 days, respectively. The obtained positivity rates for the
different combinations were correlated to the expected positivity rate (Table
4a).
Repeatability
The repeatability of the Vivalytic Noro-, Rotavirus, C. diff test was
established using a panel with 1 concentration (3x c95) of Norovirus,
Rotavirus and Clostridioides difficile. At 1 test site, the mix was tested on
the same set of Vivalytic instruments by the same operator with 3 LOTs in 20
replicates, respectively, yielding a total of 60 observations per target
pathogen. The obtained positivity rates for the different combinations were
correlated to the expected positivity rate (Table 4b).
Interferences
Interferences were evaluated for endogenous and exogenous substances, that are
potentially present in the patient sample. Refer to Table 5 for substances
that have the potential to interfere with the test.
Clinical Performance evaluation Sensitivity and Specificity
Sensitivity and specificity results derived from native liquid and soft human
stool samples. Samples were collected in a clinical setting and compared with
results of reference methods. Samples for testing with Vivalytic Noro-,
Rotavirus, C. diff cartridges were freshly used or frozen for storage and
prepared as describe above in eNAT® (COPAN Italia S.p.A.).
In case of reference testing samples were prepared according to
recommendations
of used reference methods. In total, 1 59 samples were analysed. Sensitivity
or Positive
Percent Agreement (PPA) was calculated as 100 % x TP/ (TP+FN). Specificity or
Negative
Percent Agreement was calculated as 100 % x TN / (TN+FP). The results of the
clinical
performance evaluation are shown in Table 6 Table 7 and Table 8.
Technical Support
If you require any support, technical help or have additional questions,
please contact your local distributor or visit the Bosch Vivalytic website at
www.bosch-viva lytic.com.
References
- Kuijper EJ, Coignard B, Tüll P; ESCMID Study Group for Clostridium difficile; EU Member States; European Centre for Disease Prevention and Control. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect. 2006 Oct;12 Suppl 6:2-18.
- Czepiel J, Dr6idi M, Pituch H, Kuijper EJ, Perucki W, Mielimonka A, Goldman S, Wultarhska D, Garlicki A, Biesiada G. Clostridium difficile infection: review. Eur J Clin Microbiol Infect Dis. 2019 doi: 10.1007/s10096-019-03539-6. Epub 2019 Apr 3. PMID: 30945014; PMCID: PMC6570665.
- Kordus SL, Thomas AK, Lacy DB. Clostridioides difficile toxins: mechanisms of action and antitoxin therapeutics. Nat Rev Microbiol. 2022 May;20(5):285-298.
- Gerding DN, Johnson S, Rupnik M, Aktories K. Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance. Gut Microbes. 2014 Jan-Feb;5(1):15-27.
- Lopman B, Brown D, Koopmans M: Human caliciviruses in Europe. Journal of Clinical Virology 2002; 24:137-160
- 0h D, Gaedicke G, Schreier E: Viral agents of acute gastroenteritis in German children: Prevalence and molecular diversity. J Med Virol 2003; 71:82-93
- Schneider T, Mankertz J, Jansen A., Schreier E, Zeitz M: Norovirusinfektionen — häufigste Ursache akuter Gastroenteritiden in den Wintermonaten. Deutsches Ärzteblatt 2005; 38:2551-2556
- Parashar UD, Hummelmann EG, Bresee JS et al.: Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 2003; 9:565—572
- Mas Marques A, Diedrich S, Schreier E: Group A Rotavirus Genotypes in Germany 2005/2006. Arch Virol 2007; 152:1743-1749
- Anderson EJ, Weber SG: Rotavirus infection in adults. Lancet Infect Dis 2004; 4:91—99
- ICOPAN Instructions for Use of Copan eNATE Collection and Preservation System; Version EIFU002R01 Date 2021.06.
Symbols
*Determined by using Vircell material (surrogate strain R20291 Ribotype 027 (toxin A + B positive) for a spiking approach in eNAT® (COPAN) supplemented with approximately 1000 Human Bronchial/Tracheal Epithelial Cells (hPBTEC) and 6% (v/v) Stool Matrix Negative Control (artificial simulated matrix).
**Determined by using quantified clinical isolates (Rotavirus group A,
Norovirus GII) for a spiking approach in eNAT® (COPAN) supplemented with
approximately 1000 Human Bronchial/Tracheal Epithelial Cells (hPBTEC) and 6%
(v/v) Stool Matrix Negative Control (artificial simulated matrix). Norovirus
GI was confirmed at the 3x LoD (as determined for Norovirus GII) using
artificial nucleic acid constructs.
*1 sample was detected as false negative for C. difficile.
**2 samples were detected as false positive for C. difficile
In total, 159 clinical samples were tested within the scope of the clinical study. In this data set, 52 samples were found positive for Norovirus, 54 samples were found positive for Rotavirus and 49 samples were found positive for C. difficile. 14 samples were found negative for all pathogens. All samples were tested with the two reference tests from Seegene (Allplex™ GI- Bacteria(I) and Allplex™ GI-Virus Assay) and Vivalytic Noro-, Rotavirus, C. diff to determine the clinical performance for Vivalytic. Discrepant results were verified using RIDAGENE RIDA®GENE HSP (R-Biopharm) (12 samples).
For more information see www.bosch-vivalytic.com
Read More About This Manual & Download PDF: