Enteroscreen-Wb: Rapid Test For Detection of Igm and Igg Antibodies To S. Typhi in Serum/Plasma/Whole Blood Device

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Size : 137 x 218 mm

TM

ENTEROSCREEN-WB
Rapid test for detection of IgM and IgG antibodies to S. typhi in serum/plasma/whole blood
DEVICE
INTENDED USE
ENTEROSCREEN-WBTM is a rapid, self performing, qualitative, sandwich immunoassay for the detection and differentiation of
IgM and IgG antibodies to S. typhi in human serum/plasma or whole blood specimen.

SUMMARY
Typhoid fever is a systemic prolonged febrile illness caused by a bacteria Salmonella typhi. The disease is transmitted through
ingestion of food or water contaminated with faeces or urine of infected persons.
Acute typhoid fever is characterized by prolonged fever, disturbances of bowel functions (constipation or diarrhoea),
headache, malaise and anorexia. Cough is common in the early stage of illness. Chronic carrier is determined when excretion
of S.typhi in stools or urine lasts for longer than one year after onset of acute typhoid fever. Accurate diagnosis of typhoid fever
at an early stage is not only important for etiological diagnosis but to identify and treat the potential carriers and prevent acute
typhoid fever outbreaks. Specific agglutinins appear during the course of most of the attacks during the second week of
infection. Detectable levels of IgM antibodies against S.typhi appear and persists for four months, IgG antibodies are detected
thereafter and remain in blood for two years. The detection of IgM reveals acute typhoid in the early phase of infection, while
the detection of both IgG and IgM suggests acute typhoid in the middle phase of infection. In areas of high endemicity, where
the rate of typhoid transmission is high, the detection of specific IgG increases.
In the conventional Widal test the interpretation of results is done against a baseline titre in the same geographical area since
titres of diagnostic significance differ in endemic or non-endemic areas. A paired sera with a fourfold rise in titer is needed for a
meaningful result.
The limitations of the traditional methods have prompted novel tests to be developed. ENTEROSCREEN-WBTM qualitatively
detects and differentiates between IgM and IgG class of antibodies specific to S. typhi in human serum/plasma or whole blood
specimens.

PRINCIPLE
ENTEROSCREEN-WBTM utilizes the principle of agglutination of antibodies/ antisera with respective antigen in immuno-
chromatography format along with use of nano gold particles as agglutination revealing agent. ENTEROSCREEN-WBTM isa dual
test device assembly comprising of an IgM detection test assembly and an IgG detection test assembly. The conjugate pad of
the IgM test assembly consists of two components, Agglutinating sera for Human IgM conjugated to colloidal gold and rabbit
globulin conjugated to colloidal gold. Similarly the IgG test assembly consists of Agglutinating sera for Human IgG conjugated
to colloidal gold and rabbit globulin conjugated to colloidal gold. As the test specimen flows through the respective membrane
test assemblies, the Agglutinating sera for Human IgM or the Agglutinating sera for Human IgG -colloidal gold conjugate
complexes with the S. typhi specific IgM or IgG antibodies in the specimen and travels on the membrane due to capillary
action along with the rabbit globulin-colloidal gold conjugate. This complex moves further to the test regions of the respective
test assembly where the specimen is immobilized by the S. typhi specific antigen coated at the test regions of the IgM/ IgG
device assembly leading to formation of a pink to pink- purple colored band at the test regions of the respective test devices
which indicates a positive IgM or IgG test result. The absence of this colored band in either of the test regions indicates a
negative test result.
In both the test membrane assemblies the unreacted conjugate and unbound complex, if any move further on the membranes
and are subsequently immobilized by the Agglutinating sera for rabbit globulin coated on the membranes at the control region
(C), forming a pink to pink-purple colored band. This control band acts as a procedural control and serves to validate the
results.

REAGENTS AND MATERIALS SUPPLIED


ENTEROSCREEN-WBTM kit contains:
A. Individual pouches, each containing -
1. Dual test device :
IgM Test Assembly: Membrane assembly pre-dispensed with Agglutinating sera for Human IgM - colloidal gold
DEVICE conjugate, rabbit globulin - colloidal gold conjugate, S. typhi specific antigen and Agglutinating
sera for rabbit globulin coated at the Test region ‘T’ and Control region ‘C’ respectively and
IgG Test Assembly: Membrane assembly pre-dispensed with Agglutinating sera for Human IgG - colloidal gold
DEVICE conjugate, rabbit globulin - colloidal gold conjugate, S. typhi specific antigen and Agglutinating
sera for rabbit globulin coated at the Test region ‘T’ and Control region ‘C’ respectively.
2. Desiccant pouch.
3. PIPETTE Disposable Plastic Sample Applicator.
B. BUF Sample Running Buffer in a dropper bottle.
C. Package Insert.

Colour C M Y K

Black 0 0 0 100

Green 100 20 100 10


Size : 137 x 218 mm

REF 501030010 501030025


10 25

OPTIONAL MATERIAL REQUIRED


Calibrated micropipette capable of delivering 5 µl sample accurately.

STORAGE AND STABILITY


The sealed pouches in the test kit & the kit components may be stored between 4°C to 30°C till the duration of the shelf life as
indicated on the pouch / carton. DO NOT FREEZE. After first opening of the sample running buffer bottle, it can be stored
between 4°C to 30°C for remaining duration of its shelf life.

NOTES
1. Read the instructions carefully before performing the test.
2. For in vitro diagnostic use only. NOT FOR MEDICINAL USE. For professional use only.
3. Do not use the kit beyond expiry date and do not re-use the test device.
4. Do not intermix reagents from different lots.
5. Contact with the contents of desiccant pouch containing, among other substances, cobalt chloride (CAS# 7646-79-9)
should be kept to a minimum. Inhalation / swallowing may cause harm.
6. Handle all specimens as if potentially infectious. Follow standard biosafety guidelines for handling and disposal of
potentially infectious material.
7. If desiccant colour at the point of opening the pouch has turned from blue to pink or colourless, another test device must be
run.
8. Sample running buffer contains Sodium Azide (0.1%), avoid skin contact with this reagent. Azide may react with lead and
copper in the plumbing and form highly explosive metal oxide. Flush with large volumes of water to prevent azide build-up
in the plumbing.

SPECIMEN COLLECTION AND PREPARATION


1. ENTEROSCREEN-WBTM uses human serum / plasma / whole blood as specimen.
2. No special preparation of the patient is necessary prior to specimen collection by approved techniques.
3. For whole blood, collect blood with a suitable anticoagulant such as EDTA or Heparin or Oxalate and use the freshly
collected blood.
4. Whole blood should be used immediately and should not be frozen.
5. Though fresh specimen is preferable, in case of delay in testing, it may be stored at 2°C to 8°C for maximum up to 24 hrs.
6. If serum is to be used as specimen, allow blood to clot completely. Centrifuge to obtain clear serum.
7. Repeated freezing and thawing of the specimen should be avoided.
8. Do not use turbid, lipaemic and hemolysed serum/plasma.
9. Do not use hemolysed, clotted, contaminated, viscous/turbid specimens.
10. Specimen containing precipitates or particulate matter must be centrifuged and the clear supernatant only should be used
for testing.
11. Refrigerated specimens must be brought to room temperature prior to testing.

TESTING PROCEDURE AND INTERPRETATION OF RESULTS


1. Bring the kit components of ENTEROSCREEN-WBTM device to room temperature before testing.
2. Open a foil pouch by tearing along the “notch”.
3. Remove the testing device and the sample applicator. Once opened, the device must be used immediately.
4. Label the device with specimen identity.
5. Place the testing device on a flat horizontal surface.
6. Carefully dispense 5µl of either whole blood / serum / plasma into the specimen port ‘A1’ and 5µl of same specimen into the
specimen port ‘A2’ of the test device using a micropipette OR using the 5µl sample applicator provided, dip the sample
applicator in the sample container and blot the sample in the sample port ‘A1’ and then using the same sample applicator,
dip into the sample container again and blot the sample in the sample port ‘A2’.
7. Add five drops each of sample running buffer into the buffer port ‘B1’& ‘B2’ .
8. At the end of 15 minutes, read results as follows:
Negative Result
IgM

C T A1
B1 If only one colored band appears in the Control region ‘C’ of both the test windows. It
indicates absence of antibodies to S. typhi in the specimen or that the amount of antibodies
IgG

C T A2
B2
is below detection limit of the test.

Positive Result
IgM

C T A1
B1 IgM positive
In addition to a colored band appearing in the Control region ‘C’ in both the IgM and IgG Test
IgG

C T A2
B2
windows, a colored band appears in the Test region ‘T’ of the IgM test window. The presence
Size : 137 x 218 mm

of only IgM antibodies is indicative of current acute typhoid infection. The intensity of the test
band may be more or less than the Control band, depending upon the concentration of
antibodies in specimen.
IgM and IgG positive
In addition to a colored band appearing in the Control region ‘C’ in both the IgM and IgG Test
windows, a colored band is observed in the Test region ‘T’ of both IgM and IgG Test windows.
IgM

C T A1
B1
The presence of both IgM and IgG antibodies indicates acute typhoid fever (in the middle
IgG

C T A2
B2 stage of Salmonella typhi infection). The intensity of the test band may be more or less than
the Control band, depending upon the concentration of IgM and IgG antibodies in specimen.
IgG positive
In addition to a colored band appearing in the Control region ‘C’ in both the IgM and IgG Test
windows, a colored band appears in the Test region ‘T’ of the IgG test window. The presence
IgM

C T A1
B1 of only IgG antibodies is indicative of previous Salmonella typhi infection (in which case
current fever may not be due to typhoid) or relapse or re-infection. The intensity of the test
IgG

C T A2
B2
band may be more or less than the Control band, depending upon the concentration of
antibodies in specimen.

Invalid Result
IgM

C T A1
B1
The test is invalid if the Control band in both or either one of the devices is not visible at
IgG

C T A2
B2 fifteen minutes. Verify the test procedure and repeat the test with a new device.

PERFORMANCE CHARACTERISTICS
Internal Evaluation
In an in-house study, the performance of ENTEROSCREEN-WBTM was evaluated using a panel of seventy specimens of WIDAL-
positive (of varying reactivity) and WIDAL-negative sera. The results of the evaluation are as follows:
SPECIMEN DATA WIDAL ENTEROSCREEN-WBTM COMMERCIAL RAPID TEST
No. of specimens tested 70 70 70
No. of positive specimens 16 16 16
No. of negative specimens 54 54 54
Based on this evaluation:
Sensitivity of ENTEROSCREEN-WBTM : 100%
Specificity of ENTEROSCREEN-WBTM : 100%

External Evaluation-I
Fifty samples that were Widal positive/Widal negative sera were evaluated with ENTEROSCREEN-WBTM in a reputed hospital in
Amritsar, India.The results of the evaluation are as follows:
SPECIMEN DATA WIDAL ENTEROSCREEN-WBTM
No. of specimens tested 50 50
No. of positive specimens 5 5
No. of negative specimens 45 45
Based on this evaluation:
Sensitivity of ENTEROSCREEN-WBTM :100%
Specificity of ENTEROSCREEN-WBTM :100%

LIMITATIONS OF THE TEST


1. The membrane is laminated with an adhesive tape to prevent surface evaporation. Air pockets or patches may appear,
which do not interfere with the test results. Presence of a band at the test region even if low in intensity or formation is a
positive result.
2. The deliberate slow reaction kinetics of ENTEROSCREEN-WBTM is designed to maximize and enhance reaction time
between sample capture and tracer elements to improve test sensitivity.
3. Most positive results develop within 15 minutes. However, certain sera sample may take a longer time to flow. Therefore,
negatives should be confirmed only at 30 minutes. Do not read results after 30 minutes.
4. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the result of a single test, but should only
be made by the physician after all clinical and laboratory findings have been evaluated.
5. ENTEROSCREEN-WBTM should be used as a screening test in clinically suspected cases only, and its results should be
confirmed by other supplemental method before taking clinical decisions.
Size : 137 x 218 mm

6. In some studies, it has been reported that low titre IgM antibodies to S.typhi may persist for about 4 months post infection.
Therefore, in endemic area, samples positive yet with low signal intensity should be interpreted with caution, preferably in
light of patient history.
7. The following chart would explain the IgM seroresponse in S.typhi infected subjects after onset of fever.
Detectable IgM Response
Onset of fever Percent positive
4-6 days 43.50 %
6-9 days 92.90 %
>9 days 100 %

8. A negative result, i.e., the absence of detectable IgM antibody does not rule out recent or current infection, as the positivity
is influenced by the time elapsed from the onset of fever and immunocompetence of the patient. However, if S. typhi
infection is still suspected, obtain a second specimen 5-7 days later and repeat the test.
9. High titer Rheumatoid factor may result in a false positive reaction.
10. A low extent of cross reactivity may be observed with S. paratyphi infection.

WARRANTY
This product is designed to perform as described on the label and package insert. The manufacturer disclaims any implied
warranty of use and sale for any other purpose.

BIBLIOGRAPHY
1. Hatta M et al., Simple dipstick assay for the detection of Salmonella typhi-specific IgM antibodies and the evolution of the
immune response in patients with typhoid fever. Am. J. Trop. Med. Hyg., 66(4), 2002, pp. 416-421.
2. Gopalkrishna V., Sekhar W. Y., Soo E. H., Vinsent R. A. & Devi S., Singapore Med J. 2002 Vol. 43(7) 354-358.
3. House Deborah, Wain John, Ho Vo A., Diep To S., Chinh Nguyen, Bay Phan V., Vinh Ha, Due Minh, Parry Christopher M.,
Dougan Gordon, White Nicholas J., Hien Tran Tinh & Farrar Jeremy J. J. of Clin. Microbiol. 2001 Vol. 39 (3) 1002-1007.
4. Bhutta ZA et al., Rapid Serologic Diagnosis Of Pediatric Typhoid Fever In An Endemic Area: A Prospective Comparative
Evaluation Of Two Dot-Enzyme Immunoassays And The Widal Test. Am. J. Trop. Med. Hyg. 61(4), 1999, pp. 654-657.
5. Agarwal PK et al., Typhoid Fever. JIACM 2004; 5(1):60-4.
6. Data on file: Zephyr Biomedicals.

SYMBOL KEYS
Consult
Temperature
Limitation
Instructions
for use
Date of
Manufacture 2 Do not reuse

In vitro Diagnostic Sample Running


Manufacturer IVD Medical Device
This side up BUF Buffer

Catalogue
Use by REF Number DEVICE Device
EC REP
Disposable
Contains sufficient Batch Number / Authorised Representative
for <n> tests LOT Lot Number PIPETTE Plastic Sample
Applicator
in the European Community

Manufactured by:
Zephyr Biomedicals
A Division of Tulip Diagnostics (P) Ltd.
M 46-47, Phase lIl B, Verna Industrial Estate, Verna, Goa - 403 722, INDIA.
Regd. Office: Gitanjali, Tulip Block, Dr. Antonio Do Rego Bagh, Alto Santacruz,
0817/VER-01

Bambolim Complex P.O., Goa - 403 202, INDIA.

EC REP
CMC Medical Devices & Drugs S.L., Spain.

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