AccuBioTech ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) User Manual

AccuBioTech ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) User Manual

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Catalog No. ABT-IDT-B64
Product Name Rotavirus/Adenovirus Combo Cassette (Feces)

A rapid, one step test for the qualitative detection of rotavirus and adenovirus in human feces.
For professional in vitro diagnostic use only.

INTENDED USE

ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) is a rapid chromatographic immunoassay for the qualitative detection of rotavirus and adenovirus in human feces specimens to aid in the diagnosis of rotavirus or adenovirus infection.

SUMMARY

Acute diarrhea disease in young children is a major cause of morbidity worldwide and is a leading cause of mortality in developing countries.1 Rotavirus is the most common agent responsible for acute gastroenteritis, mainly in young children.2
Its discovery in 1973 and its association with infantile gastroenteritis represented a very important advancement in the study of gastroenteritis not caused by acute bacterial infection. Rotavirus is transmitted by oral-fecal route with an incubation period of 1-3 days. Although specimen collections taken within the second and fifth day of the illness are ideal for antigen detection, the rotavirus may still be found while diarrhea continues. Rotaviral gastroenteritis may result in mortality for populations at risk such as infants, the elderly and immunocompromised patients.3
In temperate climates, rotavirus infections occur mainly in the winter months. Endemics as well as epidemics affecting some thousand people have been reported.4 With hospitalized children suffering from acute enteric disease up to 50% of the analyzed specimen were positive for rotavirus.5
The viruses replicate in the cell nucleus and tend to be host species specific producing a characteristic cytopathic effect (CPE). Because rotavirus is extremely difficult to culture, it is unusual to use isolation of the virus in diagnosing an infection. Instead, a variety of techniques have been developed to detect rotavirus in feces.

Research has shown that enteric adenoviruses, primarily Ad40 and Ad41, are a leading cause of diarrhea in many of these children, second only to the rotaviruses.6,7,8,9 These viral pathogens have been isolated throughout the world, and can cause diarrhea in children year round. Infections are most frequently seen in children less than two years of age, but have been found in patients of all ages. Further studies indicate that adenoviruses are associated with 4 – 15% of all hospitalized cases of viral gastroenteritis.5,6,7,8,9 Rapid and accurate diagnosis of gastroenteritis due to adenovirus is helpful in establishing the etiology of gastroenteritis and related patient management. Other diagnostic techniques such as electron microscopy (EM) and nucleic 36acid hybridization are expensive and labor- intensive.
With the self-limiting nature of adenovirus infection, such expensive and labor-intensive tests may not be necessary. ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) is a rapid chromatographic immunoassay for the qualitative detection of rotavirus and adenovirus in human feces specimen, providing results in 10 minutes. The test utilizes antibody specific for rotavirus and adenovirus to selectively detect rotavirus and adenovirus from human feces specimens.

PRINCIPLE

ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) is a qualitative, lateral flow immunoassay for the detection of rotavirus and adenovirus in human feces specimen.
In this test, the membrane is pre-coated with anti-rotavirus antibody on the R test line region of the test and anti-adenovirus antibody on the A test line region of the test. During testing, the specimen reacts with the particle coated with anti-rotavirus antibody and anti-adenovirus antibody. The mixture migrates upward on the membrane chromatographically by capillary action to react with anti-rotavirus antibody and anti-adenovirus antibody on the membrane and generate red line（s）. The presence of these red lines in test line region indicates a positive result, while their absence indicates a negative result. To serve as a procedural control, a red line will always appear in the control line region indicating that proper volume of specimen has been added and membrane wicking has occurred.

REAGENTS

The test contains anti-rotavirus antibody and anti-adenovirus antibody coated particles and anti-rotavirus antibody and anti-adenovirus antibody coated on the membrane.

PRECAUTIONS

• For professional in vitro diagnostic use only. Do not use after expiration date.

• The test cassette should remain in the sealed pouch until use.

• Do not eat, drink or smoke in the area where the specimens or kits are handled.

• Do not use test if pouch is damaged.

• Handle all specimens as if they contain infectious agents.
  Observe established precautions against microbiological hazards throughout testing and follow standard procedures for proper disposal of specimens.

• Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are being tested.

• The used tests, specimens and potentially contaminated materials should be discarded according to the local regulations.

• Humidity and temperature can adversely affect results.

STORAGE AND STABILITY

Store as packaged in the sealed pouches either at room temperature or refrigerated (2-30°C). The test is stable through the expiration date printed on the sealed pouch. The test must remain in the sealed pouch containing desiccant until use. DO NOT FREEZE. Do not use beyond the expiration date.

SPECIMEN COLLECTION AND PREPARATION

• Viral detection is improved by collecting the specimens at the onset of the symptoms. It has been reported that the maximum excretion of rotavirus and adenovirus in the feces of patients with gastroenteritis occurs 3-5 days after onset of symptoms. If the specimens are collected long after the onset of diarrheic symptoms, the quantity of antigen may not be sufficient to obtain a positive reaction or the antigens detected may not be linked to the diarrheic episode.
• The feces specimen must be collected in clean, dry, waterproof container containing no detergents, preservatives or transport media.
• Bring the necessary reagents to room temperature before use.

MATERIALS

Materials Provided

• Test cassettes
• Droppers
• Specimen collection tubes with extraction buffer
• Package insert

Materials Required But Not Provided

• Specimen collection containers
• Timer
• Centrifuge and pipette to dispense 80µL if required

TEST PROCEDURE

Allow the test cassette, specimen, and buffer to reach room temperature (15-30°C) prior to testing.

1. To collect fecal specimens:
   Collect sufficient quantity of feces (1-2ml or 1-2g) in a clean, dry specimen collection container to obtain enough virus particles.
   Best results will be obtained if the assay is performed within 6 hours after collection. Specimen collected may be stored for 3 days at 2-8°C if not tested within 6 hours. For long-term storage, specimens should be kept below -20°C.

2. To process fecal specimens:
   For Solid Specimens:
   Unscrew the cap of the specimen collection tube then randomly stab the specimen collection applicator into the fecal specimen in at least 3 different sites to collect approximately 50mg of feces (equivalent to 1/4 of a pea). Do not scoop the fecal specimen.
   For Liquid Specimens:
   Hold the dropper vertically, aspirate fecal specimens, and then transfer 2 drops (approximately 50µL) into the specimen collection tube containing the extraction buffer.
   Tighten the cap onto the specimen collection tube, and then shake the specimen collection tube vigorously to mix the specimen and the extraction buffer.

3. Bring the pouch to room temperature before opening it. Remove the test cassette from the foil pouch and use it within one hour.
   Best results will be obtained if the test is performed immediately after opening the foil pouch.

4. Hold the specimen collection tube upright and unscrew the tip of the specimen collection tube. Invert the specimen collection tube and transfer 2 full drops of the extracted specimen (approximately 80L) to the specimen well (S) of the test cassette, and start the timer. Avoid trapping air bubbles in the specimen well (S). See illustration below.

5. Read the results at 10 minutes after dispensing the specimen.
   Do not read results after 20 minutes.

Note: If the specimen does not migrate (presence of particles), centrifuge the extracted specimen contained in the extraction buffer vial. Collect 80µL of supernatant, dispense into the specimen well (S). Start the timer and continue from step 5 onwards in the above instructions for use.

INTERPRETATION OF RESULTS

POSITIVE:

Rotavirus   Positive: * A red line appears in the control line region (C) and another red line appears in the T2 line region.

Adenovirus Positive: * A red line appears in the control line region (C) and another red line appears in the T1 line region.

Rotavirus and Adenovirus Positive: * A red line appears in the control line region (C) and two other red lines appear in T1 line region and T2 line region respectively

NOTE: The intensity of the color in the test line region (T1/T2) will vary depending on the concentration of rotavirus or adenovirus antigens present in the specimen.
Therefore, any shade of color in the test line region (T1/T2) should be considered positive.

NEGATIVE: One red line appears in the control line region (C). No line appears in the test line region (T1/T2).

INVALID: Control line (C) fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the test with a new test cassette. If the problem persists, discontinue using the test kit immediately and contact your local distributor.

QUALITY CONTROL

An internal procedural control is included in the test. A red line appearing in the control line region (C) is an internal positive procedural control. It confirms adequate membrane wicking.
Control standards are not supplied with this kit; however, it is recommended that positive and negative controls be tested as a good laboratory practice to confirm the test procedure and to verify proper test performance.

LIMITATIONS

1. ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) is for in vitro diagnostic use only. The test should be used for the detection of human rotavirus and adenovirus in feces specimens only. Neither the quantitative value nor the rate of increase in human rotavirus and adenovirus concentration can be determined by this qualitative test.
2. ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) will only indicate the presence of rotavirus and adenovirus in the specimen and should not be used as the sole criteria for the conforming rotavirus and adenovirus to be etiological agent for diarrhea.
3. As with all diagnostic tests, all results must be interpreted together with other clinical information available to the physician.
4. If the test result is negative and clinical symptoms persist, additional testing using other clinical methods is recommended.
   A negative result does not at any time preclude the possibility of rotavirus or adenovirus infection with low concentration of virus particles.

EXPECTED VALUES

ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) has been compared with latex agglutination method, demonstrating an overall accuracy of >99.0%.

PERFORMANCE CHARACTERISTICS

Clinical Sensitivity, Specificity and Accuracy The performance of ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) has been evaluated with 605 clinical specimens collected from children and young adults in comparison with latex agglutination method. The results show that ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces) has high sensitivity and specificity for rotavirus and adenovirus.

For Rotavirus:

Negative
Positive| 191| 2| 193
Negative| 0| 168| 168
Total Results| 191| 170| 361

• Relative Sensitivity: >99.9% (98.4% – 100.0%)*
• Relative Specificity: 98.8% (95.8% – 99.9%)*
• Relative Accuracy: 99.4% (98.0% – 99.9%) m95% Confidence Intervals

For Adenovirus:

Method| Latex Agglutination| Total
Results
---|---|---
ACCU-TELL
Rotavirus/Adenovirus
Combo Cassette (Feces)| Results| Positive| Negative
Positive| 60| 1| 61
Negative| 0| 183| 183
Total Results| 60| 184| 244

Relative Sensitivity: >99.9% (95.1% – 100.0%)
Relative Specificity: 99.5% (97.0% – 100.0%)
Relative Accuracy: 99.6% (97.7% – 100.0%)
95% Confidence Intervals

Precision Intra-Assay

Within-run precision has been determined by using 15 replicates of five specimens: a negative, a rotavirus low positive, an adenovirus low positive, a rotavirus medium positive and an adenovirus medium positive. The specimens were correctly identified >99% of the time.

Inter-Assay

Between-run precision has been determined by 15 independent assays on the same five specimens: a negative, a rotavirus low positive, an adenovirus low positive, a rotavirus medium positive and an adenovirus medium positive. The specimens were correctly identified >99% of the time.

Cross-Reactivity

Cross reactivity with following organisms has been studied at 1.0×109 organisms/ml. The following organisms were found negative when tested with ACCU-TELL® Rotavirus/Adenovirus Combo Cassette (Feces).

BIBLIOGRAPHY

1. Wadell, G. Laboratory Diagnosis of Infectious Diseases: Principles and Practices. New York: Springer-Verlag, Volume II, 1988: 284-300.
2. WILHELMI I, ROMAN E, SANCHEZ-FAUQUIER A. Viruses causing gastroenteritis. Clin Microbiol Infect. April. 2003, vol.9:247-262
3. Cubitt, WD (1982) Rotavirus Infection: An Unexpected Hazard in Units Caring for the Elderly. Geriatric Medicine Today 1: 33-38
4. Hung, T et al (1984) Waterborne outbreak of Rotavirus Diarrhoea in Adults in China caused by a Novel Rotavirus. Lancet, May 26;1(8387): 1139-1142
5. Cukor, G; Perron, DM; Hudson, R and Blacklow, NR (1984) Detection of Rotavirus in Human Stools by Using Monoclonal Antibody. J. Clin. Microl. 19: 888-892
6. Wood, D. J. and A. S. Bailey. “Detection of Adenovirus Types 40 and 41 in Stool Specimens by Immune Electron Microscopy.” Journal of Medical Virology, 1987; 21: 191-199.
7. Nishio, Osamu, M. Ooseto, K. Takagi, Y. Yamasita, Y. Ishihara, and S. Isomura. “Enzyme-Linked Immunosorbent Assay Employing Monoclonal Antibodies for Direct Identification of Enteric Adenoviruses (Ad40, 41) in Feces.” Microbiol. Immunol. 1990; 34(10): 871-877.
8. Wood, D. J., K. Bijlsma, J. C. de Jong, and C. Tonkin. “Evaluation of a Commercial Monoclonal Antibody-Based Enzyme Immunoassay for Detection of Adenovirus Types 40 and 41 in Stool Specimens.” Journal of Clinical Microbiology, June 1989; 27(6): 1155-1158.
9. Thomas, Eva. E., D. Roscoe, L. Book, B. Bone, L. Browne, and V. Mah. “The Utility of Latex Agglutination Assays in the Diagnosis of Pediatric Viral Gastroenteritis.” Am. J. Clin. Pathol. 1994; 101:742-746.

GLOSSARY OF SYMBOLS

| Catalog number| | Temperature limitation
---|---|---|---
| Consult instructions for use| | Batch code
| In vitro diagnostic medical device| | Use by
| Manufacturer| | Do not reuse

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