FREQUENTLY ASKED QUESTIONS

Real-time PCR, or qPCR, is a molecular technique used to amplify and detect nucleic acids while the reaction is taking place. Fluorescent signals are monitored during each amplification cycle, allowing target DNA or RNA to be detected quickly and accurately.

In infectious disease diagnostics, real-time PCR can be used to detect DNA from microorganisms or, after a reverse transcription step, RNA from pathogens directly from suitable clinical samples.

The Ct (cycle threshold) or Cq (quantification cycle) value indicates the PCR cycle at which the amplification signal reaches a defined detection threshold.

Within the same assay and under the same conditions, a lower Ct value generally corresponds to a higher initial amount of target nucleic acid, while a higher Ct value is usually associated with a lower amount.

Ct values should always be interpreted according to the specific assay criteria and together with the performance of the assay controls.

Multiplex PCR allows several genetic targets to be detected simultaneously within a single PCR reaction.

By combining different primers, probes and fluorescence channels, one assay can detect multiple microorganisms or genetic markers at the same time.

Multiplex PCR is particularly useful for syndromic testing, where different pathogens can cause similar clinical symptoms.

VIRPLEX includes multiplex solutions for areas such as respiratory, gastrointestinal, sexually transmitted and other infectious diseases.

VIRPLEX is Vircell’s range of real-time PCR assays for the molecular diagnosis of infectious diseases.

The VIRPLEX portfolio includes assays for different diagnostic areas, including:

• Respiratory infections
• Gastrointestinal infections
• Sexually transmitted infections
• Mycobacterial infections
• Central nervous system infections
• Zoonotic and vector-borne diseases

VIRPLEX reagents are available in different formats to adapt to different laboratory workflows and sample volumes.

The extraction requirements depend on the specific VIRPLEX assay and sample type.

For workflows that require nucleic acid extraction, Vircell offers extraction solutions as well as MagXtract® 3200, which can automate nucleic acid extraction and PCR setup.

The Instructions for Use of each VIRPLEX assay should always be followed when selecting and validating the extraction procedure.

VIRPLEX assays use lyophilized reagents to support reagent stability and simplify handling and transportation.

Depending on the product format, VIRPLEX solutions may include lyophilized master mix and positive controls, as well as pre-dispensed formats designed to reduce manual preparation steps.

Depending on the assay, VIRPLEX kits are available in different formats, including vials, pre-dispensed strips (-LP) and divisible low-profile plates (-LPD).

The -LPD format allows laboratories to separate the plate into strips and use only the wells required for each PCR run.

The formats available depend on the specific VIRPLEX assay.

Yes. VIRPLEX assays can be integrated into an automated molecular workflow using MagXtract® 3200 and VIRCOM PCR.

MagXtract® 3200 automates nucleic acid extraction and PCR setup, while VIRCOM PCR supports assay configuration, analysis, interpretation and result communication.

Together, these solutions can help maintain traceability from the original sample through to the final result.

Yes. VIRCOM PCR can connect the molecular workflow to the laboratory information system (LIS).

The software supports bidirectional communication and different communication formats, helping laboratories transfer sample information and results between the molecular workflow and their LIS.

This can reduce manual data entry and improve traceability.

No. The regulatory status may vary between VIRPLEX products.

Depending on the assay and reference, products may have different regulatory classifications, including CE-IVDR, CE or RUO (Research Use Only) status.

The regulatory status and commercial availability of a specific product should always be checked on the product page and in the latest product documentation.

An internal control is included within the assay to monitor the performance of an individual sample or reaction, while an external molecular control is processed independently to monitor the performance of the assay or laboratory workflow.

Third-party external molecular controls provide an independent way to monitor analytical performance without relying exclusively on controls supplied by the assay manufacturer.

PCR inhibition occurs when substances in the clinical sample or introduced during sample processing interfere with nucleic acid extraction or amplification.

Possible inhibitors may include:

• Mucus
• Blood components
• Hemoglobin
• Heparin
• Certain transport media
• Excess cellular material
• Residues from the extraction process

An internal control can help identify potential inhibition before a result is reported.

An invalid PCR result means that the assay criteria required to reliably interpret the sample have not been met.

Possible causes include:

• PCR inhibition
• Internal control failure
• Extraction failure
• Reagent degradation
• Incorrect reaction setup
• Instrument problems
• Sample handling issues

When an invalid result occurs, laboratories should follow the troubleshooting and repeat-testing procedure defined in the assay Instructions for Use.

PCR contamination can be reduced by separating workflow stages and minimizing the transfer of nucleic acids or amplified products between samples and laboratory areas.

Common measures include:

• Unidirectional workflow
• Separation of pre-amplification and post-amplification areas
• Dedicated equipment and consumables
• Routine cleaning and decontamination
• Appropriate personal protective equipment
• Correct pipetting practices
• Negative controls in each run

Automation can also reduce manual handling during selected stages of molecular testing.

Clinical samples should be stored and transported according to the requirements of the specific assay and sample type.

Whenever possible, samples should be processed within the recommended timeframe after collection.

Temperature, transport medium, storage duration and repeated freeze-thaw cycles can affect nucleic acid integrity and PCR performance, particularly for RNA targets.

AMPLIRUN® and AMPLIRUN® TOTAL external molecular controls can be used as tools within PCR assay verification and ongoing quality monitoring processes.

AMPLIRUN® can support the evaluation of amplification and detection performance, while AMPLIRUN® TOTAL can monitor a broader workflow including nucleic acid extraction.

However, using a specific control does not by itself demonstrate compliance with ISO 15189. Each laboratory must define and document its verification and quality control strategy according to its own accreditation requirements.

Serology is the study and detection of antibodies and, in some assays, antigens in biological samples to provide information about an infection or the immune response to a microorganism.

In infectious disease diagnostics, serological tests can help identify recent or previous exposure, assess immune status and complement other diagnostic methods.

Their clinical value and interpretation depend on the microorganism, the marker being tested, the timing of sample collection and the clinical context.

Serology mainly evaluates the immune response to an infection, whereas PCR directly detects the genetic material of the microorganism.

PCR can be particularly useful when the pathogen is present in the sample and its DNA or RNA needs to be detected. Serology, on the other hand, can provide information about the antibody response that develops after exposure.

The most appropriate technique depends on the microorganism and the stage of infection. In some diseases, molecular and serological methods can be complementary.

Serum and plasma are the most commonly used samples for infectious disease serology.

However, accepted sample types depend on the specific assay. Laboratories should always check the Instructions for Use to confirm which specimen types have been validated.

Many VirClia® assays from Vircell, for example, are designed for the detection of antibodies in human serum or plasma.

No. A positive IgM result does not by itself confirm an acute or recent infection.

Although IgM may appear during the early stages of many infections, it can sometimes persist for extended periods, show cross-reactivity or produce false-positive results.

IgM results should therefore be interpreted according to the microorganism, the timing of sample collection, symptoms, epidemiological history and, when appropriate, other laboratory findings.

Not necessarily. A positive serological result means that the marker targeted by the assay has been detected, but its clinical significance depends on the type of marker and the disease.

Antibody detection may reflect a recent infection, a past infection or a previous immune response. Antigen detection assays may provide different information.

Results should always be interpreted according to the criteria of the specific test and the clinical context.

Seroconversion is the change from a seronegative to a seropositive status for a specific antibody between two different points in time.

It can be demonstrated by testing samples collected at different stages of infection. Depending on the microorganism and testing method, a significant rise in antibody titre between acute and convalescent samples may also provide useful evidence.

Seroconversion can support the diagnosis of a recent infection when this criterion is established for the specific disease.

IgG avidity measures the overall strength with which IgG antibodies bind to their target antigens.

Following a primary infection, antibody affinity can mature over time. Therefore, in certain infections, low IgG avidity may be associated with a more recent primary infection, while high avidity may be more consistent with an older exposure.

Interpretation depends on the microorganism and the assay being used.

Distinguishing a recent infection from a past infection usually requires evaluating the overall antibody pattern rather than relying on a single result.

Depending on the disease, laboratories may consider:

• IgM detection.
• IgG detection and evolution.
• Seroconversion.
• Changes in antibody titres.
• IgG avidity.
• Paired acute and convalescent samples.

These patterns should not be generalized across all infectious diseases. Interpretation must follow the specific criteria established for the microorganism and assay.

Cross-reactivity occurs when an antibody reacts with an antigen that is similar to, but different from, the antigen that originally triggered the immune response.

This can occur between closely related microorganisms that share antigenic structures and may complicate the interpretation of certain serological tests.

The relevance of cross-reactivity depends on the microorganism and assay design.

Yes. For some diseases, vaccination can generate antibodies that may later be detected by serological tests.

The significance of the result depends on the assay, the antigen being detected and the patient’s vaccination history.

Vaccination history should therefore be considered when interpreting relevant serological results.

Serological interpretation can be influenced by pre-analytical, analytical and clinical factors.

These include:

• Timing of sample collection.
• Individual immune response.
• Immunosuppression.
• Previous vaccination.
• Previous exposure to the same or related microorganisms.
• Cross-reactivity.
• Assay sensitivity and specificity.
• The antibody or antigen being detected.

Results should therefore always be interpreted according to the validated criteria of the specific assay.

The most appropriate serological technique depends on the infection being investigated and the needs of the laboratory.

CLIA can be particularly useful for automated workflows and laboratories requiring flexibility across different parameters. ELISA is a well-established format that can support batch-based processing. IFA remains valuable for certain microorganisms where antibody patterns or titres provide relevant diagnostic information.

The technique should therefore be selected according to the diagnostic application rather than the technology alone.

Vircell offers a range of immunodiagnostic technologies for infectious disease testing, designed to meet different laboratory needs and workflows.

The portfolio includes:

• VirClia®: automated CLIA immunoassays in Monotest format.
• ELISA: enzyme immunoassays for antibody detection.
• IFA: indirect immunofluorescence assays for viruses, bacteria, parasites and fungi.
• DFA: direct immunofluorescence assays.
• BRUCELLACAPT® and Rose Bengal: solutions for the serological diagnosis of brucellosis.
• VIRapid®: rapid tests for selected applications.
• External immunological controls.

Vircell has more than 30 years of experience in infectious disease diagnostics, with an in-house portfolio covering both immunodiagnostics and molecular diagnostics.

Explore the serological porfolio

Yes. Vircell offers external immunological controls designed to provide independent monitoring of selected immunodiagnostic processes.

One example is VIRCELL® ASPERGILLUS GALACTOMANNAN Ag CONTROL, an unassayed external control formulated in human serum for assays detecting Aspergillus fumigatus galactomannan antigen.

It is designed to support monitoring of sample processing and the analytical phase of galactomannan detection assays.

ELISA (enzyme-linked immunosorbent assay) is an immunoassay technique used to detect antibodies or antigens through an enzyme reaction that produces a measurable signal.

In infectious disease diagnostics, ELISA is mainly used to analyze antibody responses against different microorganisms in samples such as serum or plasma.

The technique allows multiple samples to be processed in a microplate format and can be performed manually or using automated systems. Vircell ELISA kits are specifically developed for infectious disease serology.

ELISA assays can detect different classes of antibodies, including IgG, IgM and IgA, depending on the microorganism and the diagnostic purpose of the test.

Vircell’s ELISA portfolio includes assays for different areas of infectious disease diagnostics, including respiratory infections, TORCH infections, vaccine-preventable diseases, vector-borne diseases, sexually transmitted infections, zoonoses and gastrointestinal infections.

ELISA can be particularly suitable for laboratories that want to process multiple samples using a microplate-based workflow.

The individual break-apart wells used in Vircell ELISA kits allow laboratories to adapt the number of wells used to the actual workload, while compatibility with automated ELISA systems supports different laboratory configurations.

For laboratories that need to process individual tests without waiting for a batch, a Monotest solution such as VirClia® may provide a different workflow approach.

Vircell offers a broad ELISA menu for antibody detection against microorganisms associated with different infectious diseases.

The portfolio includes assays for:

• Respiratory infections.
• TORCH infections.
• Vaccine-preventable diseases.
• Vector-borne diseases.
• Sexually transmitted infections.
• Zoonotic diseases.
• Gastrointestinal infections.
• Mononucleosis and other infectious diseases.

The available targets include microorganisms such as Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, cytomegalovirus, rubella virus, Toxoplasma gondii, Epstein-Barr virus, Helicobacter pylori and dengue virus, among others.

Vircell ELISA kits are designed so that a separate sample predilution step is not required.

When dilution or sorbent treatment is needed, it can be performed directly in the well, simplifying sample preparation and reducing additional manual steps.

μ-Capture is an ELISA format in which IgM antibodies from the patient sample are captured directly by anti-human IgM antibodies attached to the well.

This approach is used in selected IgM assays and can help reduce certain interferences associated with other antibodies present in the sample.

Vircell uses μ-Capture technology in selected IgM assays, including several TORCH and other infectious disease parameters.

Vircell provides several auxiliary components for its ELISA assays, including:

• ELISA SORBENT, for selected IgM and IgA assays.
• SERUM DILUENT, for sample dilution.
• WASH BUFFER, for washing steps.

These products are designed for use with the corresponding Vircell ELISA kits.

Yes. Vircell ELISA assays can be integrated into either manual or automated laboratory workflows.

Ready-to-use liquid reagents and individual break-apart wells help laboratories adapt each plate to the number of samples that need to be processed.

The recommended shaking speed depends on the type of shaker and should provide sufficient mixing without causing splashing or cross-contamination between wells.

As a reference, Vircell has obtained suitable results at approximately 900 rpm using a vortex-type shaker with a plate adapter and around 300 rpm using an orbital shaker.

After shaking, liquid should not remain attached to the adhesive film covering the plate. The specific assay conditions should always be checked in the Instructions for Use.

Samples should be stored according to the specimen type and the conditions specified in the Instructions for Use of the assay.

Storage temperature, duration and repeated freeze-thaw cycles can affect sample integrity and therefore the reliability of the result.

For Vircell ELISA kits, laboratories should follow the validated conditions specified for the particular assay and for serum or plasma samples.

A false-positive ELISA result can occur because of non-specific reactivity, cross-reactivity or other interference affecting the analytical reaction.

Possible causes include:

• Heterophile antibodies.
• Antibodies against related microorganisms.
• Other immunological interference.
• Non-specific reactions.
• Technical issues during processing.

The likelihood and significance of these interferences depend on the microorganism and assay design. Results should therefore be interpreted together with clinical information and other laboratory findings.

Poor reproducibility between duplicate ELISA wells usually indicates variability introduced during one or more processing steps.

Possible causes include:

• Inconsistent pipetting.
• Insufficient mixing.
• Uneven washing.
• Differences in incubation time.
• Contamination.
• Incorrect handling of the plate or reagents.

Standardizing the workflow and automating selected steps can help reduce this variability.

Quality controls help confirm that an ELISA assay has performed within the established criteria before patient sample results are interpreted.

They can help detect deviations associated with reagents, instruments, pipetting or other stages of the analytical process.

In Vircell ELISA kits, controls and samples are designed to follow equivalent processing conditions, supporting consistent assay performance.

Vircell provides technical and scientific documentation to support the selection, implementation and routine use of its ELISA assays.

Depending on the product, available resources may include:

• Instructions for Use through eIFU.
• Product information.
• Performance characteristics.
• Scientific literature.
• ELISA product flyers.
• Vircell Immunology Catalogue.
• Additional technical resources for registered users.

The latest documentation for each product reference should always be used as the primary source for specific assay requirements.

Selected Vircell ELISA kits include semi-quantitative protocols using specific calibrators.

Availability depends on the individual assay. Laboratories should consult the Instructions for Use of the specific product to confirm whether a semi-quantitative protocol is available.

VirClia® is Vircell’s range of chemiluminescence immunoassays (CLIA) in Monotest format for infectious disease diagnostics.

Each VirClia® Monotest allows an individual determination to be performed using ready-to-use reagents contained within the strip itself.

Currently, VirClia® Monotest assays are processed automatically on VirClia® Lotus, Vircell’s CLIA analyzer specifically designed for this assay range.

VirClia® Monotest assays are currently processed on VirClia® Lotus.

VirClia® Lotus is a fully automated chemiluminescence immunoassay analyzer that combines the Monotest format with random access, continuous loading and urgent sample management.

CLIA, or chemiluminescence immunoassay, is an immunodiagnostic technique that uses a light-emitting chemical reaction to detect and measure antigen-antibody interactions.

The light signal generated during the assay is measured by the analyzer and used to determine the result according to the criteria defined for each test.

VirClia® combines CLIA technology with a Monotest format developed specifically for infectious disease diagnostics.

Each VirClia® Monotest contains the reagents required to perform one individual determination, together with a calibrator and a negative control.

Each strip consists of 3 reaction wells and 5 reagent wells, which are used automatically during assay processing.

This design provides individual quality control for each Monotest.

No. VirClia® does not require a calibration curve to be generated for each routine.

The calibrator included in each Monotest forms part of the individual control of the determination, simplifying processing and avoiding the periodic calibration procedures associated with other immunoassay formats.

Most VirClia® references are supplied as kits containing 24 individual Monotests.

Some references are available in other presentations, so the specific product content should always be checked in the catalogue or product documentation.

VirClia® includes both antibody and antigen detection assays.

Depending on the reference, the range includes IgG, IgM, IgA, combined immunoglobulin assays, quantitative assays, avidity tests and antigen detection assays.

For example, VirClia® includes assays for detecting Aspergillus galactomannan antigen and Helicobacter pylori antigen, as well as a broad range of serological tests.

Yes. VirClia® Lotus allows different VirClia® parameters to be assigned to the same sample and managed within the same routine.

This facilitates serological profiles or combinations of parameters without having to process them in separate batches.

Compatibility and specific requirements depend on the individual assay.

VirClia® uses CLIA technology in a Monotest format, while ELISA typically uses a colorimetric reaction in a microplate format.

From a workflow perspective, VirClia® allows each determination to be processed individually, whereas ELISA is often particularly suitable for processing multiple samples for the same parameter in a plate-based workflow.

Vircell offers both technologies to accommodate different sample volumes, test menus and laboratory workflows.

Yes. The Monotest format allows laboratories to use only one strip for each determination that needs to be processed.

There is no need to open or use an entire plate to analyze only a small number of samples for a particular parameter.

In addition, the calibrator and negative control are integrated into each Monotest, avoiding additional consumption associated with calibration and routine control materials.

VirClia® kits generally have a shelf life of 15 months from the date of manufacture when stored under the specified conditions.

The expiry date and exact storage requirements should always be checked on the product label and in the Instructions for Use of the specific reference.

If the Monotest quality control does not meet the established acceptance criteria, the result should not be validated until the cause has been investigated.

The laboratory should review factors including:

• Product storage conditions and expiry date.
• Correct identification and loading of the sample and strip.
• Reagent condition.
• Instrument performance.
• Acceptance criteria specified in the Instructions for Use.

The assay should be repeated when required by the corresponding procedure.

The VirClia® catalogue provides the available assay menu, while Vircell’s eIFU platform gives access to the Instructions for Use and product-specific documentation.

The latest documentation for each reference should always be consulted to confirm sample types, storage conditions, assay procedure, result interpretation and regulatory status.

VirClia® Lotus is an automated chemiluminescence immunoassay (CLIA) analyzer for infectious disease diagnostics. It processes VirClia® assays in monotest format and allows laboratories to combine different samples and parameters within the same routine using random access and continuous loading.

VirClia® Lotus delivers the first results in approximately 70 minutes. After that, the system can report a new result every 35 seconds.

VirClia® Lotus can accommodate up to 50 primary sample tubes simultaneously, as well as 50 dilution tubes and 6 diluent bottles.

Once a sample has been dispensed, the system notifies the user so that the tube can be removed and the position used for a new sample.

Random access allows VirClia® Lotus to process different samples and tests independently, without waiting for a complete batch.

In practice, laboratories can combine different parameters, add new samples during the routine and prioritize urgent samples according to their needs.

VirClia® Lotus can assign up to 39 different parameters to the same sample, supporting multiparametric testing within a single routine.

Yes. VirClia® Lotus includes an integrated scanner for sample and VirClia® strip identification.

Barcode reading helps maintain traceability throughout the workflow and reduces the risk of identification errors between the sample, the requested test and the reported result.

Used VirClia® strips are automatically discarded into the instrument’s solid waste container.

The system can continue loading and processing new tests without requiring the user to remove each strip manually.

Yes. Through VirCom middleware, VirClia® Lotus can store control and calibrator results and use them for quality control monitoring.

Results can be reviewed by parameter, date and product lot, and VirCom can generate quality control charts showing mean values and the corresponding ranges.

Yes. VirClia® Lotus supports bidirectional LIS connectivity through VirCom middleware.

The system can receive sample information from the laboratory information system and send results back to the LIS once testing is completed, helping maintain traceability and reduce manual data entry.

VirClia® Lotus requires routine and preventive maintenance to support stable long-term operation.

Laboratories have access to procedures and resources for carrying out regular maintenance tasks, while Vircell Technical Service can provide specialized support whenever required.

Indirect immunofluorescence (IFA) is an immunodiagnostic technique used to detect specific antibodies against microorganisms through a fluorescent reaction.

During the assay, antibodies present in the patient sample bind to antigens fixed on a slide. A fluorescent-labelled conjugate is then added, allowing the reaction to be visualized under a fluorescence microscope.

In infectious disease diagnostics, IFA is particularly useful when the fluorescence pattern, antibody titre or comparison between different antigens provides relevant information for interpretation.

IFA remains relevant because it allows laboratories to visually assess antibody reactivity and determine antibody titres through serial dilutions.

For some diseases, these titres or fluorescence patterns provide information that cannot be obtained in the same way from a simple positive or negative result.

In specific diagnostic settings, including Q fever and some spotted fever group rickettsioses, IFA remains an important serological reference method.

An antibody titre in IFA is the highest dilution of a sample at which a specific fluorescent reaction can still be detected.

When a sample is tested through serial dilutions, the titre corresponds to the last dilution that still meets the assay criteria for positivity.

A higher titre means that reactivity remains detectable at a greater dilution, but it does not by itself confirm an active infection.

Yes. An IFA test can be negative during the early stages of infection if detectable antibody levels have not yet developed.

This is particularly relevant in infections where the serological response develops after symptom onset.

When clinical suspicion remains high, a second sample collected later or a complementary diagnostic method may be required.

Seroconversion is the change from an initially seronegative sample to a later sample in which specific antibodies are detected.

When demonstrated using samples collected at different times and interpreted according to the criteria established for the infection, seroconversion can provide evidence of a recent immune response.

Vircell offers a range of indirect immunofluorescence assays for the serological diagnosis of different infectious diseases.

The current IFA portfolio includes assays for respiratory bacterial infections, vector-borne diseases, zoonoses and fungal infections, with parameters such as Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Rickettsia conorii, Rickettsia typhi, Coxiella burnetii, Bartonella henselae, Bartonella quintana, Leishmania infantum, Trypanosoma cruzi and invasive candidiasis through CAGTA.

Vircell also offers Multitest solutions such as PNEUMOBACT and PNEUMOSLIDE.

P- references correspond to complete IFA kits, while S- references correspond to slide presentations.

P- kits provide the components required to perform the assay, whereas S- references are intended for laboratories that specifically require the corresponding substrate or antigen slide.

Vircell IFA Multitests allow antibodies against several microorganisms to be evaluated using the same slide.

Current solutions include PNEUMOBACT, which contains antigens from five bacterial agents associated with respiratory infections, and PNEUMOSLIDE, which combines bacterial and viral antigens related to respiratory infections.

This format allows several agents to be evaluated within a common workflow when differential diagnosis is required.

Microimmunofluorescence (MIF) is an immunofluorescence format that allows reactivity against several antigens to be evaluated separately in different areas of the same slide.

It is particularly useful when comparing antibody responses against related microorganisms.

Vircell uses this approach in its Chlamydophila pneumoniae assays, where different Chlamydia species are presented in separate wells to support differential interpretation.

Vircell IFA SORBENT is a reagent designed to capture human IgG and reduce interference in selected IgM or IgA IFA assays.

It is based on anti-human IgG antibodies directed against the Fc region.

It should only be used when specified in the procedure for the corresponding assay.

Coxiella burnetii has two antigenic phases, known as phase I and phase II, and the antibody response to these phases provides useful information in the diagnosis of Q fever.

In acute infection, the antibody response generally predominates against phase II antigen.

High or increasing IgG titres against phase I antigen may be associated with persistent focal infection or chronic forms of Q fever, but they should not be interpreted in isolation. Diagnosis requires clinical correlation and additional diagnostic criteria.

A single positive sample does not always confirm a recent Rickettsia infection.

Antibodies can persist following a previous infection, and cross-reactivity between related species may occur.

For some rickettsial diseases, comparing an acute sample with a convalescent sample to demonstrate a significant rise in titre provides stronger serological evidence.

Background fluorescence can occur when fluorescent signal is present but does not clearly correspond to the expected specific reaction.

Possible causes include:

• Insufficient washing.
• Residual salts.
• Excessive incubation times.
• Drying of the wells.
• Contamination.
• Non-specific sample reactivity.

When high background is observed, the laboratory should review the complete procedure and confirm that the Instructions for Use have been followed correctly.

Weak fluorescence may result from a low concentration of specific antibodies or from technical factors related to assay processing or slide reading.

The laboratory should review:

• Reagent preparation and storage.
• Incubation conditions.
• Washing procedures.
• Conjugate preparation.
• Microscope performance and settings.

Fluorescence intensity should always be interpreted according to the assay criteria and compared with the appropriate controls.

The appearance of Vircell Rickettsia and Bartonella slides may differ because of the antigen preparation process used during manufacturing.

Vircell removes residual Vero cell material used during the culture of these microorganisms.

The aim is to reduce non-specific fluorescence caused by antibodies that may react with residual cellular components and to support a more specific interpretation of the reaction.

IFA slides and reagents should be stored according to the specific conditions stated in the Instructions for Use of each product.

Incorrect storage can affect antigen stability, fluorescent conjugates and overall assay performance.

Conjugates and other light-sensitive components should be handled and stored according to the requirements of the individual reference.

Current documentation for Vircell IFA products is available through the Vircell eIFU platform and individual product pages.

Laboratories should always use the documentation corresponding to the specific product reference and lot to confirm:

• Assay procedure.
• Sample types.
• Storage conditions.
• Interpretation criteria.

In Coxiella burnetii serology, a predominant antibody response to phase II is typically associated with acute Q fever, whereas high and persistent phase I IgG titres are particularly relevant when persistent or focalized infection is suspected, such as endocarditis or vascular infection.

Phase I and phase II are two antigenic variants of Coxiella burnetii, not two consecutive stages of Q fever. Phase I is the natural, virulent form of the bacterium and has a complete lipopolysaccharide (LPS), whereas phase II has a shorter, truncated LPS and is less virulent.

Antibodies produced during acute Coxiella burnetii infection can react strongly with phase II antigen even though natural infection occurs with phase I bacteria. The complete LPS of phase I partially masks other bacterial antigens, while these antigens are more exposed in phase II because its LPS is truncated. When phase I bacteria are processed by host cells during infection, these antigens become accessible to the immune system and generate antibodies that also react with phase II antigen.

Phase II IgM and IgG antibodies typically appear during the first weeks of acute Q fever, and phase II IgG increases during convalescence. During the early course of infection, antibody titres against phase II generally reach higher levels than those against phase I, making the phase II response particularly relevant in the serological assessment of acute Coxiella burnetii infection.

Phase I IgG antibodies are particularly important when persistent or focalized Coxiella burnetii infection is suspected. Phase I IgG may also increase during recovery from acute Q fever, but high and persistent titres are especially relevant in clinical contexts such as endocarditis or vascular infection and should be interpreted together with clinical findings and changes in antibody titres over time.

Testing antibodies against both phase I and phase II improves the interpretation of Coxiella burnetii serology. Assessing both antigenic phases and monitoring how antibody titres evolve over time helps distinguish different serological patterns and provides a more complete picture of the immune response to Q fever.

Vircell’s COXIELLA BURNETII I+II IFA IgG/IgM/IgA allows antibodies against phase I and phase II Coxiella burnetii antigens to be evaluated within the same serological approach. This supports the assessment of the different antibody patterns associated with C. burnetii infection.

Molecular controls are reference materials used to verify that a nucleic acid detection assay is performing consistently and that its results can be interpreted with confidence.

Depending on the type of control, they can be used to monitor different stages of the molecular workflow, including nucleic acid extraction, amplification and detection.

They may also support method development, optimization and verification, performance studies and routine laboratory quality control programs.

An internal control is part of the assay itself and helps verify the performance of an individual sample or reaction, while an external control is processed independently to monitor the performance of the method or workflow.

Internal controls are particularly useful for identifying issues such as inhibition or failures affecting an individual reaction.

External controls provide complementary information about the overall stability and consistency of the analytical system over time.

AMPLIRUN® is Vircell’s range of molecular controls based mainly on purified microbial DNA or RNA for monitoring and evaluating the amplification and detection stages of molecular assays.

Most references contain the purified complete microbial genome, allowing different target regions of the microorganism to be evaluated.

The controls are supplied in lyophilized format and can be used for applications such as positive controls, assay optimization and verification, specificity studies, limit of detection studies and standard curve generation when appropriate.

No. AMPLIRUN® contains purified genetic material and is not designed to simulate a complete clinical sample.

For this reason, AMPLIRUN® is mainly used to monitor or evaluate the amplification and detection stages, but it cannot directly assess microorganism extraction efficiency.

To monitor extraction as well, Vircell offers AMPLIRUN® TOTAL.

Yes. AMPLIRUN® references provide concentration information to support their use in different analytical applications.

As a general reference, the current portfolio includes DNA controls commonly within the 10,000–20,000 copies/µL range and RNA controls around 12,500–20,000 copies/µL, although some references have different concentrations.

The exact concentration should always be checked in the documentation and certificate for the specific product.

Yes. AMPLIRUN® controls can be used as reference material in studies designed to evaluate the limit of detection of molecular methods.

Using an appropriate experimental design and serial dilutions, laboratories can assess assay performance at different concentrations.

The control itself does not establish the limit of detection; this must be determined through an appropriately designed and documented study.

AMPLIRUN® is supplied in lyophilized format together with an appropriate reconstitution solution.

As a general reference:

• DNA controls are reconstituted with 100 µL.
• RNA controls are reconstituted with 50 µL.

The specific Instructions for Use of the individual reference should always be followed.

AMPLIRUN® TOTAL is Vircell’s range of external molecular controls based on whole inactivated microorganisms formulated in matrices designed to mimic different clinical sample types.

Because they are processed from the beginning of the workflow, they can monitor extraction, amplification and detection.

The controls are supplied in lyophilized, single-use format and at low-positive concentrations designed to provide a more representative challenge for routine molecular workflows.

AMPLIRUN® is more appropriate when the aim is to evaluate amplification or develop and characterize an assay, while AMPLIRUN® TOTAL is more appropriate when the complete workflow from sample to result needs to be monitored.

For example:

• To evaluate specificity or amplification → AMPLIRUN®.
• To generate a standard curve → quantified AMPLIRUN®.
• To study limit of detection → AMPLIRUN® within an appropriate dilution study.
• To monitor extraction + amplification + detection → AMPLIRUN® TOTAL.
• To simulate a weak positive clinical sample in routine testing → AMPLIRUN® TOTAL.

The matrix is designed to make the control behave more similarly to a clinical sample during processing.

Vircell currently offers AMPLIRUN® TOTAL controls in matrices such as:

• Plasma.
• Swab.
• Sputum.
• Exudate.
• Urine.
• Stool.
• Cerebrospinal fluid.

The matrix used depends on the reference and the intended application of the control.

Yes. AMPLIRUN® TOTAL is supplied as a quantified low-positive control.

The quantity and units depend on the specific reference, so the Instructions for Use and lot-specific documentation should always be consulted.

The volume depends on the matrix and specific reference and currently ranges approximately from 200 µL to 1 mL.

This variation allows controls to be adapted to different extraction methods and platforms.

The exact volume should be checked in the technical documentation or catalogue for the specific product.

AMPLIRUN® TOTAL has a shelf life of up to 30 months from the date of manufacture for references that specify this condition.

The actual expiry date and storage requirements should always be checked on the product and in its Instructions for Use.

Yes. Both formats provide broad compatibility, but the appropriate control type should be selected according to how the molecular platform works.

AMPLIRUN® contains purified nucleic acid and is mainly intended for the amplification stage.

AMPLIRUN® TOTAL can be used with platforms that process samples from extraction onwards and is compatible with numerous molecular and sample-to-result systems.

The current portfolio includes compatibility with platforms such as GeneXpert®, BD MAX™, FilmArray®, ELITe InGenius®, Vivalytic, Liaison® MDX and STANDARD™ M10, among others.

Yes. AMPLIRUN® controls can support both commercial assays and laboratory-developed methods when the control is compatible with the target and intended application.

They are designed for applications including optimization, performance studies, positive control use and verification of molecular methods.

No. Using AMPLIRUN® or AMPLIRUN® TOTAL does not by itself demonstrate compliance with ISO 15189.

ISO 15189:2022 establishes quality and competence requirements for medical laboratories. External molecular controls can form part of the quality control, verification, monitoring and documentation strategies implemented within that system.

Compliance depends on the laboratory’s complete set of procedures, records and evidence, as well as the requirements of the relevant accreditation body.

No. AMPLIRUN® and AMPLIRUN® TOTAL are supplied as non-infectious materials.

AMPLIRUN® contains purified genetic material from inactivated microorganisms, while AMPLIRUN® TOTAL contains whole inactivated pathogens.

Vircell provides inactivation documentation for these materials.

No. Regulatory status depends on the specific product reference.

The current portfolio includes IVDR, CE and RUO (Research Use Only) products.

Before using a control, laboratories should check the regulatory classification, intended use and commercial availability of the specific reference in their market.

An unexpected shift in Ct values may indicate variation somewhere in the analytical process, but the Ct shift alone cannot identify the cause.

Factors that should be reviewed include:

• Extraction performance when using AMPLIRUN® TOTAL.
• Pipetting and reaction preparation.
• Reagent condition.
• Lot changes.
• Storage conditions.
• Instrument performance.
• PCR configuration and analysis.
• Overall workflow variability.

The trend should be evaluated against the acceptance criteria established by the laboratory for that specific control and assay.

AMPLIRUN® TOTAL can be processed repeatedly over time to assess whether a reagent lot change is associated with a meaningful change in molecular workflow performance.

Using a consistent external material allows laboratories to compare trends across different runs, lots or time periods.

The criteria used to define a significant variation should be established within the laboratory’s quality control program.

Yes. Vircell’s molecular portfolio includes negative matrices for selected applications and can also support the development of customized matrices.

Current matrix options include products related to stool, respiratory swabs and sputum.

Availability depends on the specific application and product.

A cell line is a population of cells maintained under controlled laboratory conditions that can be used as a biological system for the culture and study of microorganisms, particularly viruses.

In clinical and experimental virology, cell lines can be used for viral isolation and propagation, infectivity studies, method development, antigen production, research applications and quality control procedures.

The appropriate cell line always depends on the microorganism, the intended application and the laboratory’s validated procedure.

The appropriate cell line should be selected according to its susceptibility to the virus of interest and the specific purpose of the laboratory procedure.

Not all viruses replicate equally well in every cell line. Selection may depend on the microorganism being cultured, the subsequent detection method, the culture format and the laboratory’s validated conditions.

Vircell provides different cell lines to support a range of clinical virology applications.

Vircell controls the quality of its cell lines through contamination testing, microscopic evaluation of cell morphology and routine monitoring throughout the production process.

Quality controls include Mycoplasma detection using specific PCR and Hoechst staining, testing for bacterial and fungal contamination, and microscopic assessment of cell morphology.

Vircell also periodically renews its working cultures from cells stored in liquid nitrogen to help limit changes associated with prolonged cell culture.

Cell culture contamination can be reduced through appropriate aseptic technique, careful workflow organization and regular monitoring of cultures.

Key measures include:

• Using aseptic procedures when handling cultures.
• Keeping biological safety cabinets and work surfaces clean and properly maintained.
• Avoiding the sharing of reagents or materials between cultures when cross-contamination is possible.
• Correctly identifying cell lines and reagents.
• Routinely monitoring cultures for contamination.
• Separating suspicious cultures from other cell lines.

A cell culture suspected of contamination should not be used until the cause has been investigated.

Poor cell growth can result from problems related to the cells themselves, reagents, culture conditions or microbial contamination.

Possible causes include:

• High passage number.
• Unsuitable culture medium or supplements.
• Incorrect temperature, atmosphere or pH.
• Overconfluence.
• Microbial contamination.
• Problems during cell recovery or handling.
• Changes in incubation conditions.
• Deteriorated media or reagents.

When poor growth is observed, both the culture history and laboratory conditions should be reviewed systematically.

Biosafety measures should be determined through a risk assessment that considers both the cell line and the microorganisms and procedures used in the laboratory.

The required containment level should not be determined solely by the name of the cell line. The origin of the material, the microorganism being inoculated, the procedures performed and the potential generation of aerosols should also be considered.

Laboratories should apply biosafety procedures, personal protective equipment, decontamination methods and waste management practices appropriate to their activities and applicable regulations.

BRUCELLACAPT® is Vircell’s immunocapture-agglutination assay for the detection of total anti-Brucella antibodies in human serum.

It is designed for the serological diagnosis of human brucellosis and is particularly useful in evolved or chronic stages of infection and for treatment follow-up.

BRUCELLACAPT® can be used in both screening and titration protocols and provides a visual result after incubation.

BRUCELLACAPT® detects total anti-Brucella antibodies, including both agglutinating and non-agglutinating or incomplete antibodies.

This broader antibody detection is one of the main characteristics of the immunocapture-agglutination method and can be particularly relevant in later stages of brucellosis.

BRUCELLACAPT® is not designed to separately report individual immunoglobulin classes such as IgG, IgM or IgA; other serological techniques should be used when class-specific information is required.

Blocking or non-agglutinating antibodies are anti-Brucella antibodies that may not produce a strong reaction in conventional agglutination tests even though they are present in the patient’s serum.

These antibodies can become particularly relevant in prolonged or chronic forms of brucellosis, where conventional agglutination methods may underestimate the serological response.

BRUCELLACAPT® is designed to detect both agglutinating and non-agglutinating antibodies, providing a broader assessment of the anti-Brucella antibody response.

Yes. BRUCELLACAPT® is particularly recommended for the serological evaluation of chronic or evolved forms of brucellosis.

In these situations, conventional agglutination methods may be less informative because the antibody profile can include non-agglutinating antibodies that BRUCELLACAPT® is designed to detect.

Serology should nevertheless be interpreted together with the clinical presentation, epidemiological exposure and other laboratory findings, particularly in focal or complicated disease.

Yes. BRUCELLACAPT® can be used to monitor changes in anti-Brucella antibody titres during patient follow-up.

Published studies have shown that a progressive decrease in BRUCELLACAPT® titres, together with clinical improvement, can support the assessment of a favourable response to treatment.

However, antibody titres may remain elevated for some time after recovery, so a persistent positive titre should not by itself be interpreted as treatment failure or active disease.

Yes. BRUCELLACAPT® supports both screening and titration protocols using the same assay format.

The BRUCAPT reference provides 96 tests when used for screening or 24 tests when used for titration.

The kit contains sufficient diluent for the titration protocol. When BRUCELLACAPT® is used in screening mode, additional SERUM DILUENT FOR BRUCELLACAPT® (Ref. B0002) is required.

IVDR is Regulation (EU) 2017/746, the European Union regulatory framework for in vitro diagnostic medical devices (IVDs). It has applied since 26 May 2022 and replaced the previous In Vitro Diagnostic Medical Devices Directive (IVDD 98/79/EC).

IVDR establishes requirements covering the safety and performance of IVDs throughout their lifecycle, including risk classification, performance evaluation, clinical evidence, traceability, conformity assessment and post-market surveillance.

Certain legacy devices can still benefit from transitional provisions under specific conditions.

IVDR classifies in vitro diagnostic medical devices into four risk classes—A, B, C and D—according to their intended purpose and the risks associated with an incorrect result.

Class A generally covers the lowest-risk devices, while Class D covers the highest-risk IVDs. Classes B and C represent intermediate levels of risk.

Classification is determined using the rules in Annex VIII of Regulation (EU) 2017/746. Most Class B, C and D devices require conformity assessment involving a notified body, while non-sterile Class A devices can generally be self-declared by the manufacturer.

CE marking on an IVD indicates that the manufacturer has demonstrated conformity with the applicable European regulatory requirements for the device and its intended purpose.

Under IVDR, the conformity assessment depends on the device classification. For devices requiring notified body involvement, the CE marking is accompanied by the identification number of the notified body.

However, a CE-marked IVD currently available in Europe is not necessarily already certified under IVDR, because eligible legacy devices may still be legally marketed under IVDD transitional provisions. The product-specific regulatory documentation should therefore always be checked.

IVDR is already applicable, but eligible legacy IVDs may continue to be placed on the European market during extended transition periods if all applicable conditions are met.

The current transition end dates are:

• 31 December 2027: Class D devices and devices covered by certain notified body certificates issued under IVDD.
• 31 December 2028: Class C devices.
• 31 December 2029: Class B devices and Class A devices placed on the market in sterile condition.

These periods apply only to eligible legacy devices that fulfil the conditions established by Regulation (EU) 2024/1860. New IVDs and non-sterile Class A devices have had to comply with IVDR since 26 May 2022.

The regulatory status of a Vircell product should be checked for the specific product reference using the latest product documentation and Vircell’s current product catalogue.

Vircell identifies regulatory status reference by reference. The current portfolio includes products already marked as IVDR, products covered by other CE regulatory status and RUO products, depending on the reference.

For this reason, laboratories and distributors should not assume that an entire Vircell product family has the same regulatory classification. Vircell’s online product catalogue and current documentation should be used to confirm the status of each product.

IVDR and ISO 15189 address different parts of the diagnostic laboratory environment: IVDR regulates in vitro diagnostic medical devices in the European Union, while ISO 15189:2022 specifies requirements for the quality and competence of medical laboratories.

A laboratory accredited to ISO 15189 may use IVDR-compliant IVDs as part of its quality-managed workflow, but IVDR compliance of a product does not make a laboratory ISO 15189 compliant, and ISO 15189 accreditation does not replace IVDR requirements.

The two frameworks therefore complement each other but should not be treated as equivalent.