Real-Time PCR

Real Time PCR is a molecular diagnostic technique used to detect and amplify nucleic acids from pathogens during the amplification process in real time. Unlike conventional PCR, Real Time PCR allows continuous monitoring of amplification through fluorescent signals, enabling faster and more precise detection.

The technique is widely used in infectious disease diagnostics because it offers high analytical sensitivity and analytical specificity, while significantly reducing turnaround time compared to traditional culture methods.

Real Time PCR can detect viral, bacterial, fungal and parasitic targets directly from clinical samples, even when present at low concentrations.

The technology also supports multiplex testing, allowing simultaneous detection of multiple pathogens within a single reaction.

Reverse Transcription PCR, is a variation of PCR used to detect RNA targets. Since PCR amplification requires DNA, viral or cellular RNA must first be converted into complementary DNA, also known as cDNA, using a reverse transcription step before amplification.

RT-PCR is commonly used for the detection of RNA viruses such as influenza, RSV, SARS-CoV-2 and many other respiratory pathogens. The technique combines high sensitivity with rapid turnaround times, making it one of the most important tools in molecular infectious disease diagnostics.

In routine laboratory workflows, RT-PCR assays are frequently integrated into multiplex syndromic panels for simultaneous detection of several respiratory or gastrointestinal pathogens.

The Ct value, or cycle threshold, represents the number of amplification cycles required for the fluorescent signal to exceed the detection threshold during a PCR run.

Lower Ct values are generally associated with higher concentrations of target nucleic acid, while higher Ct values may indicate lower pathogen loads or low-level target detection. Ct values should always be interpreted within the clinical context and according to assay-specific recommendations.

Ct values are influenced by several factors, including:

  • Sample quality
  • Extraction efficiency
  • Assay design
  • Instrument performance
  • Time of sample collection

Because Ct values are method-dependent, direct comparison between different assays or instruments is not always appropriate.

Multiplex PCR is a molecular technique that allows simultaneous detection of multiple genetic targets within a single reaction.

By combining several primer and probe sets in one assay, multiplex PCR helps laboratories improve workflow efficiency, reduce reagent consumption and shorten turnaround time. This approach is particularly valuable in syndromic testing, where multiple pathogens may cause similar clinical symptoms.

Examples include:

  • Respiratory pathogen panels
  • Gastrointestinal pathogen panels
  • Sexually transmitted infection panels

Multiplex PCR also helps optimize sample usage, especially when limited specimen volume is available.

Qualitative PCR determines whether a target nucleic acid is detected or not detected in a sample. The result is generally reported as positive, negative or invalid.

Quantitative PCR, also known as qPCR, measures the amount of target nucleic acid present in the sample. Results are typically expressed as copies/mL, IU/mL or another standardized quantitative unit.

Qualitative assays are commonly used for routine pathogen detection, while quantitative assays are frequently used for:

  • Viral load monitoring
  • Treatment follow-up
  • Disease progression assessment
  • Therapy response evaluation

Compatible sample types depend on the assay and target pathogen. Common clinical specimens used in molecular diagnostics include:

  • Nasopharyngeal swabs
  • Oropharyngeal swabs
  • Sputum
  • Bronchoalveolar lavage
  • Serum
  • Plasma
  • Whole blood
  • Stool
  • Urine
  • Genital swabs

Proper sample collection and transport are essential to preserve nucleic acid integrity and ensure reliable results.

Laboratories should always follow the assay Instructions for Use regarding validated sample types and transport conditions.

PCR reagents and molecular controls should be stored according to the conditions specified in the Instructions for Use (IFU) to preserve assay stability and performance.

General recommendations may include:

  • Storing reagents frozen or refrigerated as indicated in the IFU
  • Minimizing freeze-thaw cycles
  • Protecting fluorescent reagents from prolonged light exposure
  • Avoiding repeated temperature fluctuations
  • Monitoring freezer and refrigerator temperatures routinely

Improper reagent storage may affect amplification efficiency, Ct consistency and overall assay reliability.

Proper sample storage is critical for maintaining nucleic acid stability and ensuring accurate molecular results.

Whenever possible, samples should be processed promptly after collection. If immediate testing is not possible, specimens should be stored according to the assay recommendations and the type of sample collected.

Repeated freeze-thaw cycles should be minimized, as they may degrade nucleic acids and affect assay performance.

Transport conditions, storage temperature and transport media can all influence PCR result quality.

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

Common inhibitors include:

  • Mucus
  • Blood components
  • Hemoglobin
  • Heparin
  • Certain transport media
  • Excess cellular material

Inhibited samples may generate false negative or invalid results. Internal controls are essential for identifying inhibition events and ensuring result reliability.

Appropriate sample preparation and validated extraction procedures help minimize inhibition risk.

An internal control is a non-target material included in the PCR workflow to monitor assay performance and identify potential extraction or amplification failures.

Internal controls help laboratories detect:

  • PCR inhibition
  • Extraction failure
  • Reagent issues
  • Workflow errors

Depending on assay design, internal controls may be added during extraction or directly into the amplification reaction.

The use of internal controls is considered essential in molecular diagnostics to ensure result validity and improve workflow reliability.

Contamination prevention is one of the most critical aspects of molecular diagnostic workflows.

Recommended measures include:

  • Unidirectional workflow organization
  • Physical separation of pre- and post-amplification areas
  • Use of dedicated equipment and consumables
  • Routine environmental cleaning
  • Appropriate PPE usage
  • Regular contamination monitoring
  • Inclusion of negative controls in every run

Amplicon contamination can lead to false positive results and compromise laboratory reliability. Strict contamination control procedures are essential for maintaining assay integrity.

Before implementation into routine use, laboratories should verify assay performance according to their quality management system and accreditation requirements.

Verification studies commonly include evaluation of:

  • Precision
  • Reproducibility
  • Analytical sensitivity
  • Analytical specificity
  • Accuracy
  • Limit of detection
  • Carryover
  • Reference performance comparison

The extent of verification depends on:

  • Assay complexity
  • Intended use
  • Laboratory workflow
  • Regulatory status

All studies should be documented and reviewed according to laboratory accreditation procedures.

External molecular controls are standardized materials used to monitor assay performance independently from kit-integrated controls. They help laboratories verify that the molecular workflow is performing correctly and consistently over time.

Vircell’s AmpliRun® controls can be used as external amplification controls to monitor PCR assay performance, Ct consistency and inter-run variability.

AmpliRun® Total controls are designed to support monitoring of the complete molecular workflow, including extraction and amplification steps, making them useful for identifying failures that may occur before the PCR reaction itself.

External controls help laboratories:

  • Verify assay consistency
  • Monitor Ct variability
  • Detect workflow deviations
  • Support assay verification
  • Evaluate extraction and amplification performance
  • Maintain long-term quality assurance

They are particularly valuable for laboratories working under ISO 15189 or similar quality standards, where documented control strategies, trend monitoring and performance verification are essential.

Late Ct values may occur for several reasons, including:

  • Low target concentration
  • Poor sample quality
  • Degraded nucleic acids
  • Inefficient extraction
  • Partial inhibition
  • Incorrect sample collection timing

Late amplification should always be interpreted carefully within the clinical and technical context.

Laboratories should review:

  • Internal control performance
  • Sample integrity
  • Instrument performance
  • Extraction workflow
  • Assay-specific interpretation criteria

Invalid PCR results generally indicate that assay quality controls or internal controls did not perform as expected.

Possible causes include:

  • PCR inhibition
  • Extraction failure
  • Reagent degradation
  • Instrument issues
  • Workflow errors
  • Incorrect sample handling

When invalid results occur, laboratories should repeat testing according to internal procedures and review all quality control parameters before result release.

Available documentation may include:

  • Instructions for Use
  • Performance evaluation summaries
  • Analytical sensitivity data
  • Specificity studies
  • Precision studies
  • Verification support materials

Laboratories should evaluate assay performance according to their internal quality management system and applicable accreditation standards.

External molecular controls such as AmpliRun® and AmpliRun® Total can support accredited molecular workflows by helping laboratories monitor assay consistency, extraction performance and long-term quality assurance according to ISO 15189 quality management practices.

ELISA

ELISA, or Enzyme-Linked Immunosorbent Assay, is an immunological technique used to detect antibodies or antigens in clinical samples through specific antigen-antibody interactions.

The method combines enzymatic reactions with colorimetric detection to generate measurable signals proportional to the presence of the target analyte.

ELISA assays are widely used in infectious disease diagnostics because they provide:

  • high analytical reliability

  • scalability

  • compatibility with automated workflows

  • cost-effective routine testing

ELISA testing is commonly used for:

  • serological screening

  • immune status evaluation

  • infectious disease diagnosis

  • epidemiological studies

IgM antibodies are generally associated with the early immune response following initial exposure to an infectious agent, while IgG antibodies usually appear later and may persist long term after infection or vaccination.

In many infectious diseases:

  • IgM detection may suggest recent or acute infection

  • IgG detection may indicate past exposure or immune memory

However, interpretation depends on the specific pathogen, patient clinical history, timing of sample collection, vaccination status, immune condition of the patient

Serological results should always be interpreted together with clinical findings and additional laboratory information when necessary.

Compatible sample types depend on the specific assay and target analyte. The specimens compatible with the Vircell ELISA include serum and plasma.

Proper sample collection, transport and storage are essential to ensure accurate serological results.

Laboratories should always follow the assay Instructions for Use regarding:

  • validated sample types

  • anticoagulant compatibility

  • storage conditions

  • sample stability recommendations

Proper sample storage is important to preserve antibody and antigen stability and ensure reliable assay performance.

General recommendations include:

  • refrigeration at 2–8°C for short-term storage

  • freezing for long-term preservation

  • minimizing repeated freeze-thaw cycles

  • avoiding prolonged room temperature exposure

Improper storage conditions may affect sample integrity and contribute to inaccurate or inconsistent results.

Laboratories should always follow the assay-specific Instructions for Use and internal laboratory procedures.

Yes. Many Vircell ELISA kits are compatible with open automated ELISA processors and laboratory automation systems.

Automation may help laboratories to increase throughput, reduce hands-on time, improve reproducibility, standardize workflow and reduce manual pipetting variability

Compatibility depends on the analyzer configuration and assay requirements. Laboratories should verify and validate automation compatibility according to their workflow and instrumentation.

Proper shaking conditions are important to ensure homogeneous reagent distribution and optimal assay performance during incubation steps.

Laboratories should use orbital microplate shakers validated for ELISA applications and follow the conditions specified in the assay Instructions for Use.

Excessive shaking may increase background signal or splashing between wells, while insufficient agitation may affect reaction consistency and assay reproducibility.

Consistent incubation conditions help improve precision and reduce variability between runs. It is recommended to set a sufficiently high speed to ensure mixing of the components without spilling the contents of the wells (after shaking, there should be no liquid residue on the adhesive film covering the plate).

Borderline or equivocal ELISA results should be interpreted carefully within the clinical and laboratory context.

Borderline results may occur due to:

  • low antibody concentrations

  • early seroconversion

  • nonspecific reactivity

  • sample variability

  • cross-reactive antibodies

Depending on the clinical situation, laboratories or clinicians may consider:

  • repeat testing

  • follow-up sample collection

  • complementary diagnostic methods

  • clinical correlation

Interpretation should always follow assay-specific recommendations and laboratory procedures.

False positive serological results may occur when antibodies react non-specifically with assay components or cross-reactive antigens.

Potential causes may include cross-reactive antibodies, autoimmune conditions, heterophile antibodies, recent infections, nonspecific immune activation and technical workflow deviations

Appropriate quality control procedures, assay validation and clinical correlation are essential for accurate interpretation of serological results.

Laboratories should verify assay performance according to their quality management system and accreditation requirements.

Verification studies may include precision evaluation, reproducibility assessment, comparison studies, analytical performance review, workflow verification and quality control assessment

The extent of verification depends on:

  • intended use

  • assay complexity

  • laboratory workflow

  • accreditation requirements

All verification activities should be documented according to laboratory procedures.

Quality controls help laboratories monitor assay consistency and verify proper test performance.

Routine QC procedures include positive controls, negative controls, cut-off controls and internal laboratory quality materials

Quality control monitoring helps:

  • detect workflow deviations

  • identify reagent instability

  • improve result reliability

  • support accreditation compliance

Laboratories should follow the assay Instructions for Use and internal quality management procedures for QC implementation and documentation.

High background signal may occur when nonspecific reactions or inadequate washing interfere with assay performance.

Common causes include:

  • insufficient washing

  • incorrect reagent preparation

  • incubation deviations

  • contamination

  • excessive conjugate concentration

  • inadequate blocking conditions

High background may reduce assay specificity and complicate result interpretation.

Routine maintenance of washers, proper workflow organization and adherence to assay procedures help minimize background issues.

Poor reproducibility between duplicate wells may result from inconsistencies during sample preparation or assay handling.

Potential causes include:

  • pipetting variability

  • inadequate washing

  • uneven incubation conditions

  • improper plate handling

  • reagent instability

  • insufficient mixing

Consistent laboratory technique, calibrated equipment and standardized workflow procedures are essential for improving assay reproducibility.

ELISA reagents should be stored according to the conditions specified in the Instructions for Use to preserve assay stability and analytical performance.

General recommendations may include:

  • maintaining recommended refrigeration conditions

  • minimizing prolonged room temperature exposure

  • protecting light-sensitive reagents

  • monitoring storage temperatures routinely

Improper storage conditions may affect enzyme activity, signal generation and overall assay reliability.

Workflow optimization may be improved by:

  • organizing sample preparation consistently

  • standardizing incubation timing

  • validating washing procedures

  • monitoring reagent stability

  • implementing routine quality control review

  • training laboratory personnel regularly

Proper workflow organization helps improve reproducibility, reduce errors and maintain consistent assay performance in routine diagnostic testing.

VirClia Monotest

Chemiluminescence immunoassay (CLIA) is an immunological detection technique that uses light-emitting chemical reactions to identify antigen-antibody interactions.

CLIA technology combines high analytical sensitivity with automated workflow capabilities, making it widely used in infectious disease diagnostics and routine clinical laboratories.

Compared to conventional colorimetric immunoassays, chemiluminescence may provide:

  • enhanced analytical sensitivity

  • broader dynamic range

  • improved automation

  • faster turnaround time

Monotest technology is an assay format where each determination is individually packaged and processed independently rather than in large batches.

This approach allows laboratories to run only the required number of tests, helping reduce reagent waste and improve workflow flexibility.

Monotest systems are particularly useful for laboratories processing:

  • low daily sample volumes

  • urgent samples

  • variable workloads

  • multiple parameters simultaneously

A VirClia kit contains 24 single-use tests. Each strip is composed of 3 reaction wells and 5 reagent wells, which are used during the assay.

Additionally, some VirClia references are available in plate format, consisting of 48 tests packaged in individual plates, each composed of 12 strips. This format helps improve the processing workflow for references with higher sample volumes.

VirClia kits have a shelf life of 15 months from the date of manufacture, provided that the storage conditions are respected (2–8 °C).

VirClia systems combine chemiluminescence sensitivity with the operational flexibility of monotest technology.

The systems are designed to support:

  • random-access testing

  • reduced reagent waste

  • simplified workflow

  • rapid turnaround time

  • flexible parameter management

  • low hands-on time

VirClia analyzers are especially valuable in laboratories requiring efficient infectious disease testing without the limitations associated with traditional batch analyzers.

Random-access testing allows samples and parameters to be processed independently without waiting for completion of a full analytical batch.

This workflow improves laboratory flexibility by enabling:

  • urgent sample prioritization

  • continuous loading

  • simultaneous parameter testing

  • improved turnaround time

Random-access systems are particularly beneficial for laboratories managing unpredictable sample volumes or time-sensitive requests.

Yes. VirClia analyzers support random-access operation, allowing urgent samples to be introduced and processed immediately without interrupting routine workflow.

This capability helps laboratories improve turnaround times for priority samples and optimize daily workflow management.

VirClia monotest systems are designed to simplify workflow operation and minimize routine calibration complexity compared to some traditional batch methodologies.

Specific calibration and quality control procedures depend on the assay and analyzer configuration and should always follow the corresponding Instructions for Use.

VirClia reagents should be stored according to the conditions specified in the Instructions for Use to preserve stability and analytical performance.

General recommendations include:

  • maintaining recommended refrigeration conditions

  • avoiding temperature fluctuations

  • protecting reagents from contamination

  • monitoring storage temperatures routinely

Improper storage conditions may affect signal stability and assay reliability.

VirClia analyzers are designed to support laboratory information system (LIS) integration to facilitate sample traceability, result management and workflow standardization through a middleware.

Connectivity capabilities may depend on the LIS software and local laboratory infrastructure.

Yes. VirClia systems are designed to support simultaneous management of different infectious disease parameters within the same workflow.

This flexibility may help laboratories optimize routine operation and improve workflow efficiency.

When quality controls fall outside the expected range, laboratories should investigate the possible causes before releasing patient results.

Possible causes may include:

  • reagent degradation
  • incorrect storage conditions
  • analyzer maintenance issues
  • calibration deviations

Corrective actions should be carried out by authorized personnel who are properly trained in the technical handling of the analyzers.

VirClia assays intended for clinical diagnostic use are developed and manufactured according to applicable European regulatory requirements for in vitro diagnostic medical devices.

Product regulatory status and intended use are specified in the corresponding Instructions for Use and product documentation.

Routine maintenance is essential to ensure optimal analyzer performance and long-term workflow reliability.

General recommendations may include:

  • daily cleaning procedures

  • periodic maintenance verification

  • proper waste management

  • monitoring consumable status

  • following preventive maintenance schedules

Laboratories should always follow the analyzer-specific maintenance procedures described in the corresponding technical documentation.

Workflow optimization may be improved by:

  • organizing routine and urgent sample management

  • monitoring reagent utilization

  • implementing regular QC review

  • maintaining preventive maintenance schedules

  • training laboratory personnel appropriately

Proper workflow organization helps maximize analyzer efficiency, reduce downtime and improve turnaround times.

VirClia Lotus

VirClia Lotus is an automated chemiluminescence immunoassay platform designed for infectious disease diagnostics using monotest technology and random-access workflow.

The system is intended to provide flexible routine operation while helping laboratories optimize turnaround times, reagent utilization and workflow organization. Its design supports continuous sample loading and simultaneous parameter management according to routine laboratory needs.

VirClia Lotus processes and reports the first result in approximately 1 hour and 10 minutes. After that, a new result is reported every 35 seconds.

VirClia Lotus can process 40 tests in 1 hour and 30 minutes, and 80 tests in 2 hours and 50 minutes.

VirClia Lotus can hold up to 50 sample tubes simultaneously in the sample carousel. Once the samples have been dispensed, the instrument notifies the user and allows the tubes to be removed so that new samples can be loaded.

VirClia Lotus supports continuous loading of both samples and reagents.

VirClia Lotus allows to assign up to 39 different parameters to be tested for the same sample.

Yes. Urgent samples can be loaded at any time, even while VirClia Lotus is already in operation. The instrument allows the user to load the urgent sample and program the required test or tests as soon as possible.

The urgent sample and the requested tests are then processed immediately, without waiting for the previously programmed routine to finish. All tests requested for the urgent sample are processed simultaneously, and the results are reported consecutively.

No. VirClia Lotus uses metallic tips for dispensing samples and reagents.

The tips are washed internally and externally with a decontamination solution and wash solution, helping to prevent cross-contamination between samples and reagents.

VirClia Lotus can accommodate different types of sample tubes, including primary tubes, laboratory tubes, and pediatric cups.

VirClia Lotus also includes an adapter for 2 mL conical-bottom tubes, ensuring proper alignment to facilitate sample aspiration.

Processed strips are automatically discarded into a waste container. This keeps VirClia Lotus ready to process new tests in continuous loading mode, without direct user intervention.

Yes. Through the Vircom Middleware software, VirClia Lotus stores all results obtained in relative light units (RLUs) for negative controls and calibrators.

These results can be filtered by parameter, date, and product lot. Vircom can also generate graphs showing the mean value and the corresponding ±3 SD ranges.

Random-access testing allows laboratories to process samples independently without waiting for completion of a full analytical batch.

This workflow improves operational flexibility by allowing urgent samples to be introduced at any moment during routine operation. It may also help reduce turnaround times and improve workflow organization in laboratories with variable or unpredictable workloads.

Continuous loading allows samples, reagents and consumables to be introduced into the analyzer while routine testing is ongoing.

This functionality helps laboratories maintain uninterrupted workflow operation and optimize daily sample management without needing to stop or restart analytical runs.

Yes. VirClia Lotus is designed to support random-access operation, allowing urgent or STAT samples to be processed without waiting for completion of routine testing batches.

This capability may help laboratories improve response times for priority testing requests and optimize routine workflow management.

Yes. VirClia Lotus is designed to support simultaneous management of multiple infectious disease parameters within the same workflow.

This flexibility helps laboratories optimize routine operation and improve efficiency when processing different assays during the same analytical session.

Routine maintenance is essential to ensure optimal analyzer performance and long-term workflow reliability.

Laboratories should follow the preventive maintenance procedures specified in the system manual, including routine cleaning (daily, weekly and monthly), monitoring of consumables and periodic verification of analyzer performance.

Consistent maintenance practices help and support stable routine operation.

VirClia Lotus is designed to support laboratory information system integration, through the VirCom middleware, to facilitate sample traceability, workflow standardization and result management.

Connectivity capabilities may depend on the analyzer configuration and local laboratory infrastructure.

Indirect Immunofluorescence Assay - IFA

Indirect immunofluorescence assay is a serological method used to detect specific antibodies in patient samples. During the assay, antibodies present in the sample bind to antigens fixed onto a slide. A fluorescent-labelled conjugate is then added, allowing the reaction to be visualized under a fluorescence microscope.

IFA is commonly used in infectious disease diagnostics because it combines high sensitivity with visual interpretation. It is particularly useful for infections where antibody patterns, titers or serological evolution provide relevant diagnostic information.

IFA is considered a reference method for several infectious diseases because it allows direct visualization of specific antibody reactivity and provides information beyond a simple positive or negative result.

The technique is especially valuable when antibody titers, paired sera or phase-specific responses are clinically relevant. This is the case for several vector-borne, zoonotic and atypical infections, where interpretation often requires correlation between laboratory findings, clinical presentation and epidemiological context.

Antibody titers are determined by identifying the highest serum dilution at which specific fluorescence remains detectable. In general, higher titers indicate a stronger antibody response, although the clinical significance depends on the pathogen, timing of sample collection and patient context.

A single titer may provide useful information, but interpretation is often stronger when paired sera are available. A significant increase in titer between acute and convalescent samples may support recent or active infection.

Seroconversion is the development of detectable specific antibodies in a patient who was previously seronegative. In infectious disease serology, this may be observed when an initial sample is negative and a later sample becomes positive.

Seroconversion can provide strong evidence of recent infection, especially when interpreted together with compatible clinical findings and appropriate timing of sample collection.

Paired sera interpretation compares antibody titers from two samples collected at different stages of infection, usually an acute-phase sample and a convalescent-phase sample.

A significant increase in antibody titer between both samples may support recent or active infection. This approach is particularly useful when early serology is negative or inconclusive, since antibody production may not be detectable during the first days of infection.

Phase I and phase II antibodies are mainly relevant in Coxiella burnetii serology. These antibody responses can provide useful information about the stage or form of infection.

In general, phase II antibodies are more commonly associated with acute infection, while phase I antibodies may be associated with chronic or persistent infection. Interpretation should always consider clinical history, antibody titers, follow-up results and validated diagnostic criteria.

Nonspecific fluorescence occurs when fluorescence is observed but is not clearly related to the expected antigen-antibody reaction. It may be caused by sample quality issues, cross-reactive antibodies, inadequate washing, excessive background staining or slide drying artifacts.

Because nonspecific fluorescence can complicate interpretation, IFA results should be read by trained personnel using appropriate controls and standardized reading criteria.

Autofluorescence is natural fluorescence emitted by certain biological materials or slide components independently of the specific assay reaction. It may generate background signal or patterns that interfere with interpretation.

Correct microscope settings, appropriate controls and experienced reading help distinguish autofluorescence from true specific fluorescence.

Weak fluorescence may occur when antibody concentration is low, reagents have not been handled correctly, incubation conditions are suboptimal or microscope settings are not appropriate. It may also result from excessive washing or prolonged exposure of fluorescent reagents to light.

When weak fluorescence is observed, laboratories should review reagent storage, incubation conditions, conjugate preparation, washing steps and microscope performance before interpreting the result.

IFA slides and reagents should be stored according to the conditions specified in the Instructions for Use to preserve antigen stability and fluorescence performance.

Slides should be protected from humidity and temperature fluctuations, while fluorescent conjugates should be protected from prolonged light exposure. Incorrect storage may reduce signal intensity, increase background fluorescence or affect assay reliability.

Fluorescence microscope maintenance is essential for reliable IFA interpretation. Optical components should be kept clean, illumination performance should be monitored and filters should be checked according to the manufacturer’s recommendations.

Regular maintenance helps ensure consistent fluorescence visualization, reduces interpretation variability and supports long-term assay performance.

If the washing procedure is performed correctly, antigen detachment should not occur. If no cells or fluorescence are observed, the washing procedure should be reviewed to ensure that no step has damaged or detached the antigen from the slide.

The following points should be checked:

  • The wash solution should be prepared and stored according to the Instructions for Use.
  • Only the wash solution included in the Vircell kit should be used. Wash solutions from other manufacturers should not be used.
  • The washing times indicated in the Instructions for Use should be strictly followed.
  • Direct application of a PBS or water jet onto the well should be avoided.
  • Slide overlapping during the washing procedure should also be avoided.

The appearance of Rickettsia and Bartonella slides may differ from other commercial IFA products due to differences in antigen preparation.

As part of our production process, Vircell removes Vero cell remnants from the Rickettsia and Bartonella preparations. This is intended to improve assay specificity by reducing nonspecific fluorescence that may arise when patient serum reacts with residual Vero cell material.

By minimizing these cellular remnants, the slides are designed to reduce the risk of fluorescence patterns that could be misinterpreted as positive reactions. This antigen preparation approach supports clearer interpretation and contributes to the specificity of the assay.

If background fluorescence is observed, the procedure should be reviewed to confirm that the Instructions for Use have been followed correctly.

The following points should be checked:

  • The wash solution should be prepared and stored according to the Instructions for Use.
  • The washing protocol should be carefully reviewed. The specified washing times should be followed, and gentle agitation should be applied to ensure uniform washing of all wells.
  • The slides should be rinsed with distilled water after each wash, as residual salts from the wash solution may contribute to background fluorescence.
  • Incubation times should be verified, as longer incubation than recommended may increase background fluorescence.
  • The wells should not be allowed to dry during incubation. This is particularly important if background fluorescence is observed only in some wells. The use of a humid chamber during incubation is recommended to help prevent complete drying of the wells.

This pattern may be related to drying of the conjugate or anti-human globulin during the assay. To help prevent this, sample and conjugate incubations should be performed in a humid chamber, as indicated in the Instructions for Use.

The washing step should also be reviewed to confirm that it has been performed correctly and uniformly across all wells.

In rare cases, this type of result may also be associated with a prozone effect. However, this is uncommon and is generally observed only in sera with very high antibody titers.

When a high number of positive results is observed at the recommended screening dilution, the prevalence of the disease in the tested population or geographic area should be considered.

For example, results detected at low titers, such as 1:64, may in some cases reflect past exposure or previous infection rather than recent or active infection. In these situations, antibody titration may be necessary to support a more accurate interpretation.

Results should always be interpreted together with the clinical findings, epidemiological context and, when appropriate, follow-up serology.

When using the microimmunofluorescence (MIF) technique, the antibody response to Chlamydia species should be evaluated simultaneously against the different species included in the assay.

This approach helps assess possible cross-reactivity and supports interpretation based on the differential antibody response observed for each species. Therefore, the slide includes wells for Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila psittaci.

The kit may be used to evaluate antibody responses to any of the included species, according to the Instructions for Use. The positive control is reactive for the three species.