OnSite Typhoid IgG/IgM Rapid TestSimplifying Diagnostics of S. typhi & paratyphi Infection

Contents S. typhi and paratyphi infection Current detection methods OnSite Typhi IgG/IgM Rapid Test To be aware of

S. typhi and paratyphi Infection

• Gram negative

• Facultative rod-shaped bacterium

• Enterobacteriaceae family

S. Typhi and Paratyphi Type A, B, C

Structure of Typhi Bacterium

• O and H antigen are the most predominant antigens which induce antibody production during infection

• IgM to O antigen is seen in primary infection and re-infection • H antigen is a strong IgG immunogen

Flagellar “H” antigen

Somatic “O” antigen

S. Typhi & Paratyphi Transmission

Contaminated food or water

• Raw food

• Poor water sanitation

• Traditional sanitary style

• Inappropriate food & water preparation or storage

Traveler disease!

High Prevalence of Typhi and Paratyphi in Developing Countries

In 2000, Typhi and Paratyphi cause

• Over 2.16 million episodes worldwide

• 216,000 deaths

• More than 90% occurred in Asia

S. Typhi & Paratyphi Infection Trend

• S. Typhi infection decreases dramatically• Paratyphi infection is predominant now

Detection of both S. typhi and paratyphi are critical

Typhi & Paratyphi Fever Symptoms

• Daily increase of fatigue• Headache• Diarrhea• Malaise• Anorexia• Transient macular rash of rose-colored

spots on body trunk• Daily increase in fever of 38.5 to ℃

40 by the 3℃ rd or 4th day of illness• Constipation or diarrhea• Enlarged lymph nodes, spleen and liver

To Be Differentiated from

Present with similar symptoms (diarrhea, vomiting, fever, nausea or share similar transmission route (contaminated water source): • Malaria • Norovirus • Cholera • Influenza • Filariasis • Rotavirus • Leptospirosis• Dengue fever

Disease Course

• 6-30 day incubation • Last for one month if untreated • Serious complications: - Intestinal Hemorrhage - Intestinal Perforation• Long term carrier “Typhi Mary”

Early and accurate diagnosis is critical:• Facilitate proper treatment for early recovery and prevent deaths

• Prevent spread

Current Diagnostic Methods

Current Diagnostic Methods • Culture

- Blood culture - Bone marrow culture - Stool culture

• Serological Ab Detection

- Agglutination (widal test)

- ELISA (Typhi dot)- Lateral flow chromatographic

immunoassay (OnSite rapid test)

• Molecular Dx - Not clinically practical

Typhi Ag

A 3+ day procedure. Not a screening testAntibiotic administration reduces detection rate

Culture Detection

Widal Test: the Most Over Used Test

• Add O, H, AH, BH Widal Test reagents to all of the tubes• Incubate at 37-C overnight or rock by hands for 1 minute

• A test method developed in1896• Pre-dilute specimen to a series of dilutions

Old method Prone to operator error Time consuming Require skillful technician Cross reaction A dilution series with each target O, H, AH and BH

Not recommended by WHO

Typical Problems with Widal Test

Cross reaction with non-Typhi Salmonella or malaria

Cross reactivity with other LPS of enterobacteriaceae

Operator error

False Positive False Negative Inadequate application of

bacterial antigen on test tubes or slides

Variability in preparation of commercial antigens

Operator error

Low reproduce-abilityUnreliable with less sensitivity and specificity

Typhi Dot IgM or IgG Test

Dot EIA for the detection of IgM and IgG antibodies against a specific antigen for Salmonella typhi.

One hour long, 6 test procedures

79% sensitivity and 89% specificity

Does not detect Abs to S. paratyphi

Product needs to be refrigerated

12 month shelf life only

No built-in procedure control

Limited to S. Typhi, no indication of infection stage

Tubex IgM Test

Multiple proceduresExpensiveDoes not detect Paratyphi IgMDoes not detect IgG antibodies in chronic or relapsing patient

On market in the last ten years but not popular

OnSite Typhoid IgG/IgM Rapid Test

First Choice for Screening of Typhi and Paratyphi FeverTrusted by Pathologists of 50 countries

Identify Different Stages of Infection

IgM :Develops in 5-7 days, Declines in one month IgG: Rises in 10-14 days Last for months

specimen

capillary flow

conjugating Pad Control LineTest Line1sample Pad

Detection of anti typhi IgM

Test Line2

IgGIgM

Ag

Immobilized anti human IgM

Ag Ag

Ag-gold conjugateRabbit IgG gold (control)

specimen

IgGIgM

Reagent to bind human IgG

Ag

ImmobilizedAnti rabbit IgG

Detection of anti typhi IgG

control

OnSite Rapid Test Principle

OnSite Typhoid IgG/IgM Rapid Test

Qualitatively detects and differentiates IgG & IgM to S.typhi and paratyphi in: • Serum• Plasma• whole blood

Utilizes O & H antigens that do not present in healthy individuals

OnSite Typhoid IgG/IgM Test “1-2” Easy Steps

Step 1: 1 drop of specimen

Step 2: 1 drop of sample diluent

Step 3: Read result

OnSite Typhoid IgG/IgM Rapid Test

Negative IgM Positive Invalid

No Infection Early primary Active primary, Late stage or, infection repeat infection latent infection Re-Test

Test Result Interpretation

IgG Positive IgG/IgM Positive

OnSite Typhoid IgG/IgM Test Cross Reactivity

No cross reactivity to the diseases with similar symptoms

H. Pylori Rotavirus Leptospirosis Dengue fever

Cholera Influenza Malaria Pneumonia

Clinical study for IgM Test

OnSite Typhoid IgG/IgM Rapid Test

ELISA Positive Negative Total

Positive 31 3 34

Negative 2 198 200

Total 33 201 234

Relative sensitivity: 91.2%

Relative specificity: 99%

Overall agreement: 97.9%

OnSite Typhoid IgG/IgM Rapid Test

ELISA Positive Negative Total

Positive 13 1 14

Negative 2 198 200

Total 15 199 214

Relative sensitivity: 92.9%

Relative specificity: 99%

Overall agreement: 98.5%

Clinical study for IgG

OnSite Typhoid IgG/IgM Rapid Test Performance

OnSite Typhoid IgG/IgM Rapid Test Evaluation

Country China Indonesia Madagascar Pakistan Zimbabwe

Specimens 11 Typhi 9 Paratyphi A 60 Typhi-Paratyphi 20 Suspicious

specimens 20 suspicious specimens 138 specimens

Reference test kit

Test kit Blood culture Widal test Widal test Tubex IgM test Blood culture

Result 100% Positive 69% Positive (H) 20% Positive (H) 100% Positive12 positive, 126 negative

OnSIte Typhoid IgG/IgM Rapid Test 95% Positive 88% IgG Positive 30% IgG Positive 100% positive via

extended protocol100% sensitivity 93.7% specificity

Distributed to more than 50 countries

Test Detection Procedure Test time Cost

OnSite IgG/IgM Test

IgG and IgM to S. typhi & S.paratyphi O & H antigens

2 simple procedures

15 min Low

Widal Test Antibody to S. typhi & S. paratyphi O & H antigens

Dilution,Incubation

2 min – > 18 hrs

Low

Typhi dot Test

IgM and IgG to S. typhi OMP antigen

6 procedures 60 min Low

Culture Live bacterial replication Incubation 2-3 days High

OnSite Typhiod IgG/IgM Rapid TestThe 1st choice

Widal Test vs OnSite Rapid Test Test Parameter Widal Test OnSIte Typhoid IgG/IgM Rapid Test

Principle Agglutination immunoassay Lateral flow immunoassay

Antigen used S. typhi & S.paratyphi O and H antigens S.tphi & S.paratyphi O and H antigens

Test procedure 1. Dilute specimen to a series of 8 dilutions2. Add O, H, AH, BH reagents to all of the 8 tubes3. Incubate at 37-C overnight

1. Add specimen to the test device2. Add Sample Diluent to the test device

Result reading Visually observe sediments Visually observe colored IgG or IgM band in 15 minutes

Requirement - Dedicated lab staff- Tubes, water bath or incubator

- No equipment required- Minimal training

Sensitivity - 63.2 % sensitivity to cultureCite: J. Clinical and Diagnostic Research: 6(2):

198:199, 2012- 95%-100% sensitivity to culture

Comments - Time consuming- Prone to operator error- Losing its relevance in western and European

nations- Cross reactive- Required be stored at 2-8◦C

· Fast & inexpensive- Stored in a wide range of temperatures- High reproducibility- Can use whole blood specimen

Co-relation

Widal Test Typhi O antigen 1: 40: OnSite IgM Negative Widal Test Typhi O antigen 1: 80: OnSite IgM PositiveWidal Test Paratyohi H antigen 1: 80: OnSite IgG positive Widal Test Paratyohi H antigen 1: 160: OnSite IgG positive * Study in Madacascar, 2011

OnSite: Practical Advantages- DRUQDetection Advantages Detects antibodies to S.typhi & paratyphi A ， B ， C Differentiates IgG from IgM in the same test device Easily accessible specimens serum, plasma, whole blood, suitable for emergency care

Result Advantages Result in 15 minutes, enabling doctor to take immediate action Highly accurate & reproducible: Performance is verified in multiple regions across the world The most reliable test on the market User Advantages Can be performed by health professionals with minimal training No equipment no electricity is required 24 month stability at a wild range of temperatureQuality Advantages Launched since 2005, millions of tests are sold in more than 50 countries Key raw materials made in house in compliance with ISO 13484 to

ensure product quality and delivery quality

Final Message to Doctors

For fast, reliable and simple screening of Typhi & Paratyphi fever

Rely On Onsite Typhoid IgG/IgM Rapid tests (R0160C, R0161C)

• #1 brand of typhi IgG/IgM rapid test• Established in 2007 • Widely accepted in over 50 countries• Passed MOH evaluation studies and

registered in many countries

Together, we grow CTK

QUALITY . INNOVATION . SIMPLICITY

To Be Aware Of

To Be Aware Of #1

• Without symptoms IgG or IgM positive = Infection or Exposure

• With symptoms IgG or IgM positive = Disease

To Be Aware Of #2

Typhi is an acute infection. It progresses rapidly. Test result at initial test is negative, but can turn to positive few days late

Re-sampling is necessary if symptoms persist

Detectable level

• Proper action to be followed – Verify by confirmatory tests to make final

diagnostic decision – Conduct antibiotic sensitivity test to select proper

antibiotic for effective treatment

To Be Aware Of #3

Together, we grow CTK

QUALITY . INNOVATION . SIMPLICITY

The Common Typhi Tests on Market Test

Parameter Blood Culture Widal Test Typhoi Dot OnSIte Typhoid IgG/IgM Rapid Test

Principle Live bacterial replication Agglutination immunoassay Enzyme Immunoassay Lateral flow immunoassay

Antigen used N/A S. typhi & S.paratyphi O and H antigens S. typhi OMP antigen S.tphi & S.paratyphi O and H

antigens

Test procedure 1. Culture blood in broth from pro-enrichment

2. Dual selection enrichment

3. Dual plating. Total 3-4 day procedure

1. Pre-dilute specimen to a series of 8 dilutions

2. Add O, H, AH, BH Widal Test reagents to all of the 8 tubes

3. Incubate at 37-C overnight

1. Add 4 drops of specimens

2. Incubate for 10 mins3. Wash4. Incubate HRP-anti

human IgG or IgM 5. Wash 6. Add TMB

1. Add specimen to the test device

2. Add Sample Diluent to the test device

Result reading Traditional or biochemical identification Visually observe sediments Visually observe IgG or

IgM color dotVisually observe colored IgG or IgM band in 15 minutes

Requirement- Incubator- Electricity- Trained personnel

- Dedicated lab staff- Tubes, water bath or

incubator- Minimal training- Refrigerator

- No equipment required- Minimal training

Comments - Gold Standard method- Not a screening test

- Time consuming- Prone to operator error- Losing its relevance in

western and European nations

- Cross reactive

- 1 hour long, 7 step test procedure

- Does not detect Paratyphi

- Test components must be stored at 2-8°C

- No built-in procedure control

· Fast & inexpensive- Can be used and stored

in a wide range of temperatures

- Detects both S. typhi & Paratyphi infections

- Detects latent or chronic infection

- High reproducibility