SOP-S-004 — Dengue Duo (NS1 Ag and IgM/IgG) and Salmonella typhi (IgM/IgG) Rapid Immunoassay SOP

1.0 Purpose

This procedure covers the rapid qualitative detection and differentiation of Dengue virus NS1 antigen and IgM/IgG antibodies, and Salmonella typhi IgM/IgG antibodies, at Tarangnan Community Hospital, including procedural control checks and statutory reporting.

2.0 Scope

This procedure applies to all qualitative lateral-flow testing by Medical Technologists in the Clinical Laboratory for patients with acute febrile illness suspected of Dengue fever or Typhoid fever across inpatient, pediatric, and outpatient services. It covers specimen preparation, device operation, diagnostic interpretation, and mandatory public health disease surveillance reporting under RA 11332. General serological operations are governed by Serology Section Workflow Quality Control and Result Reporting Sop.

3.0 Definitions

• Dengue NS1 Antigen: Non-structural glycoprotein 1 synthesized by dengue virus, secreted in high concentrations into serum during the early acute viremic phase of infection (days 1 to 5).
• Dengue IgM / IgG: Specific immunoglobulins directed against dengue virus structural antigens; IgM indicates acute primary or early secondary infection, while IgG indicates past exposure or secondary immune response.
• Salmonella typhi IgM / IgG: Antibodies directed against specific lipopolysaccharide and flagellar antigens of Salmonella enterica serovar Typhi; IgM marks acute typhoid fever, and IgG marks convalescence or reinfection.
• Lateral-Flow Immunochromatography: Membrane-based assay wherein specimen migrates across antibody-gold conjugates and immobilized capture lines by capillary action.
• Procedural Control (C Line): Mandatory internal reagent line that must develop color to confirm adequate fluid migration and active conjugate chemistry.

4.0 Responsibility

4.1 Hospital Director

• Approves this procedure and ensures a continuous supply of rapid test kits and disease surveillance resources.

4.2 Pathologist

• Provides clinical and technical oversight of febrile serology testing, indeterminate findings, and disease surveillance.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Maintains version control of febrile panel SOPs, monitors reagent inventory and expiration, and audits daily QC performance.
• Ensures all testing technologists have verified competency on both Dengue and Typhoid rapid test platforms.

4.4 Medical Technologists

• Verify specimen identity, volume, and suitability before test loading.
• Perform assays strictly according to method-specific specimen volumes, buffer additions, and timing windows.
• Validate internal procedural control lines on every test device, record lot numbers, and report acute infections under RA 11332.

4.5 Laboratory Aides

• Clean and sanitize bench surfaces, record daily refrigerator temperatures, and assist in maintaining clean inventory.
• Shall not load test cassettes, dispense reagents, interpret result lines, or release laboratory findings.

5.0 Policy / Procedure

5.1 Clinical Application and Diagnostic Principles

• Febrile differential panel utility: acute Dengue and Typhoid fevers share overlapping clinical presentations during early illness in the Philippines. Rapid immunoassay testing provides diagnostic discrimination to guide patient triage and clinical management.
• Dengue serological kinetics: - NS1 Antigen: appears on Day 1 of fever and persists through Days 5–9. Optimal early acute marker before antibody seroconversion. - IgM Antibodies: detectable starting Days 3–5, peaking at 2–3 weeks, and declining over 2–3 months. - IgG Antibodies: in primary infection, IgG appears slowly by Day 10–14 and persists for life. In secondary infection, IgG rises rapidly within 1–2 days of fever onset to high titers.
• Typhoid serological kinetics: - IgM Antibodies: emerge during the first week of fever (Days 4–7), indicating acute primary typhoid infection. - IgG Antibodies: emerge in the second to third week of illness and persist, indicating convalescence, past infection, or reinfection.

5.2 Specimen Requirements and Preparation

• Acceptable specimen types include serum, plasma (heparin, EDTA, or citrate), or fresh whole blood. Serum is the preferred specimen.
• For serum collection, draw blood into plain red-top or gold-top SST tubes. Allow complete clotting for 30–60 minutes, then centrifuge at 1,000–1,200 × g for 10 minutes.
• Inspect specimen integrity: use clear, non-hemolyzed serum. Reject grossly hemolyzed, icteric, or lipemic specimens per Phlebotomy Workflow Unacceptable Specimens and Adverse Reactions Sop.
• Allow refrigerated specimens, buffer bottles, and foil-wrapped cassettes to reach ambient room temperature (15–30 °C) before testing.

5.3 Method A: Dengue Duo (NS1 Ag + IgM/IgG) Rapid Immunoassay

• Open the STANDARD Q Dengue Duo pouch. Place the dual-cassette device on a clean, level surface. Label with the patient laboratory accession number.
• Perform the Dengue NS1 antigen procedure: - Use the supplied 100 µL disposable dropper. - Dispense exactly 100 µL of serum, plasma, or whole blood into the round NS1 sample well (S). - Do not add chase buffer to the NS1 well. - Start digital timer immediately. - Read the NS1 result at 15 to 20 minutes. Do not interpret after 20 minutes.
• Perform the Dengue IgM/IgG antibody procedure: - Use the supplied 10 µL calibrated EZ capillary dropper or micropipette. - Collect 10 µL of serum, plasma, or whole blood and dispense into the IgM/IgG sample well (S). - Holding the assay buffer bottle vertically, add exactly 3 drops (approximately 90 µL) of buffer into the round buffer well. - Start digital timer immediately. - Read the IgM/IgG result at 15 to 20 minutes. Do not interpret after 20 minutes.
• Interpret Dengue Duo test lines: - NS1 Reactive: colored bands appear at both control line (C) and test line (T) on the NS1 cassette, indicating acute dengue viremia. - NS1 Non-Reactive: colored band appears only at the control line (C) on the NS1 cassette. - IgM Reactive: colored bands appear at control line (C) and IgM line (M), indicating acute primary or recent dengue infection. - IgG Reactive: colored bands appear at control line (C) and IgG line (G), indicating past exposure or secondary infection. - IgM & IgG Reactive: colored bands appear at C, M, and G lines, indicating active secondary dengue infection. - Non-Reactive (Antibody): colored band appears only at control line (C). - Invalid Result: absence of the control line (C) on either cassette invalidates that specific test. Retest using a fresh device.

5.4 Method B: Salmonella typhi (Typhoid IgM/IgG) Rapid Immunoassay

• Remove the Typhoid rapid test cassette from its sealed foil pouch. Place horizontally on the bench and label with the patient accession number.
• Load specimen and assay buffer: - Using a calibrated micropipette or the supplied micro-dropper, dispense exactly 10 µL of serum, plasma, or whole blood into the sample well (S). - Holding the Typhoid buffer dropper vertically, immediately add exactly 2 drops (approximately 60–80 µL) of Typhoid assay buffer into the sample well or dedicated buffer well per kit IFU.
• Start digital timer immediately. Read and record results at 15 to 20 minutes. Do not interpret results after 20 minutes, as non-specific background precipitation can create false bands.
• Interpret Typhoid test lines: - IgM Reactive: two colored lines appear: control line (C) and IgM line (M). Indicates acute primary typhoid fever. - IgG Reactive: two colored lines appear: control line (C) and IgG line (G). Indicates past infection, carrier state, or convalescence. - IgM & IgG Reactive: three colored lines appear: C line, M line, and G line. Indicates mid-stage acute infection or reinfection. - Non-Reactive: only the control line (C) appears. - Invalid Result: no control line (C) develops. The test is void and must be repeated with a fresh cassette.

5.5 Operational Comparison Table

Technologists shall consult this table to verify volumes and buffer additions before testing:

5.6 Quality Control and Method Limitations

• Internal control validation: the development of a distinct procedural C line is mandatory on every test window. If any C line fails to develop, the test is invalid and cannot be reported.
• External QC testing: test external positive and negative controls on every new shipment and every new lot number before releasing patient results.
• Note cross-reactivity and clinical limitations: - Dengue serology may exhibit cross-reactivity with other flaviviruses (such as Zika virus or Japanese Encephalitis). - Typhoid serology may cross-react with non-typhoidal Salmonella or other Enterobacteriaceae. - Non-reactive results during early fever (Days 1–3) do not exclude infection; if clinical suspicion persists, request a convalescent specimen in 3–5 days.

5.7 Result Reporting and Statutory Disease Notification

• Report each biomarker using standardized phrasing: - "Dengue NS1 Antigen: Non-Reactive / Reactive" - "Dengue IgM Antibody: Non-Reactive / Reactive" - "Dengue IgG Antibody: Non-Reactive / Reactive" - "Typhoid IgM Antibody: Non-Reactive / Reactive" - "Typhoid IgG Antibody: Non-Reactive / Reactive"
• Include the standard report disclaimer: "Rapid qualitative screening immunoassay. Negative results early in fever do not rule out infection. Results must be correlated clinically with complete blood count and clinical course."
• Execute mandatory public health notifications under RA 11332: - Dengue fever and Typhoid fever are Category I/II nationally notifiable infectious diseases under Republic Act No. 11332. - Immediately enter reactive cases into the Laboratory Infectious Disease Notification Logbook. - Transmit official electronic or written case reports to the TCH Hospital Epidemiology and Surveillance Unit (HESU) and Infection Prevention and Control Committee (IPCC) within 24 hours of result release.

6.0 Records

7.0 References

• DOH Clinical Laboratory QMS Tool, Section 3 — Service Delivery
• DOH Administrative Order No. 2021-0037, Annex B1 — Technical Standards for Clinical Laboratories
• ISO 15189:2022, Section 7.3 — Examination Processes
• WHO Guidelines for Dengue Diagnosis, Treatment, Prevention and Control
• Republic Act No. 11332, Mandatory Reporting of Notifiable Diseases and Health Events of Public Health Concern Act

Companion policies:

• Serology Section Workflow Quality Control and Result Reporting Sop — Serology section general workflow, QC, and result reporting
• Laboratory Reporting Policy — Result reporting, urgent notification, and clinical communication
• Laboratory Retention Schedule Policy — Master retention schedule for serological records and disease reports