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Example 3

Diagnostic Testing in Parallel

Parallel testing involves conducting 2 or more tests on an animal or group of animals. If any of the tests are positive, the animal is considered to be affected. Therefore, the second test is usually applied to the animals that were negative from the first diagnostic test. Often parallel testing is used in emergency situations where a quick decision is necessary where 2 or more tests may be applied simultaneously. Parallel testing increases sensitivity and the negative predictive value, but reduces the specificity and the positive predicitive value. Use of parallel testing essentially asks the animal to 'prove' it is healthy. Parallel testing is useful when there is an important penalty for missing a disease (false negatives) (Thrusfield, M, 1995).

Table Of Contents

  1. Example Of Testing In Parallel
  2. Salmonellosis In Horses?
  3. An Example Of Testing In Parallel Using Salmonellosis
  4. Johne's Disease In Cattle
  5. References

Example Of Testing In Parallel

If you need to review sensitivity, specificity and predictive values, please refer to the introductory section of this website. Suppose we have a quick antibody-based screening test (Test A) for a particular disease with a sensitivity of 65% and a specificity of 90%. Another antibody-based test (Test B) that is available, but more expensive, is used when you want to increase the sensitivity (sensitivity=90%; specificity=90%). For the purpose of this illustration, the total number of animals tested is 1,000 and the prevalence of this particular disease is 10%. The example is as follows:

  Disease + Disease - Total Predicted value
Test A + 65 90 155 PV+ = 41.9%
Test A - 35 810 845 PV- = 95.9%
Total 100 900 1,000  

From this initial screening test, the predictive value of a positive test was 65/155=41.9% and the negative predictive value was 810/845=95.9%. We would then incorporate the new test for those samples that were negative using the screening test and the results are as follows:

  Disease + Disease - Total
Test B + 31 81 112
Test B - 4 729 733
Total 35 810 845

The second test has resulted in an additional 31 true positive animals and 81 false positive animals. After completion of the second test, the final result of the two tests for this particular disease would be as follows:

  Disease + Disease - Total Predicted value
Test A & B + 96 171 267 PV+ = 36%
Test A & B - 4 729 733 PV- = 99.5%
Total 100 900 1,000  

After both tests have been completed, the overall sensitivity has increased to 96/100=96% and the overall specificity has decreased to 729/900=81%. In addition, the positive predictive value of the test process (41.9%) has been reduced to 36% with the overall testing of these samples. In contrast, the negative predictive value has increased from 95.9% to 99.5%. We thus have more confidence in the value of a negative test and less confidence in the value of a positive test.

Depending on the nature of the testing modalities used, the change in positive predictive value may not occur. For example, where the main test or the comparative test is detection of antigen, such as culture, the specificity is 100% and, therefore, the positive predictive value will remain at 100%, regardless of how many times you repeat the test. However, the sensitivity will increase. The following examples will demonstrate this concept.

Salmonellosis In Horses?

Salmonellosis is an enteric disease caused by Salmonella spp bacteria. These bacteria are gram negatives which may cause severe bacteremia or septicemia and which produce toxins from death of the bacteria. These bacteria may affect any species and may cause several different syndromes depending on the species affected. The range of clinical signs may vary from asymptomatic carriers to acute colitis with severe endotoxemia, sometimes leading to death of the patient. At the present time there are over 2,200 serotypes of Salmonella spp bacteria known. The definitive diagnosis of salmonellosis is accomplished by microbiologic culture of feces, tissues or blood.

In the equine species, it is very difficult to culture Salmonella spp bacteria, even in the presence of severe diarrhea. Therefore, it is recommended to obtain at least 3-5 fecal samples to increase the chance of culturing the organism. We refer to this multiple sampling as Testing in Parallel.

An Example Of Testing In Parallel Using Salmonellosis

The prevalence of Salmonella spp infections in horses that are hospitalized in the United States is reported to be approximately 65%. The sensitivity of the fecal culture test on the first sample is 55% and the specificity is 100%. The total number of horses tested in this example is 100.

  Disease + Disease - Total
Fecal Culture + 36 0 36
Fecal Culture - 29 35 64
Total 65 35 100

In the above example, the sensitivity is 55%, the predictive value of a positive test is 36/36=100% and the negative predictive value is 35/64=54.7%. We then will retest the horses that are fecal culture negative with a second fecal culture.

When testing horses for detection of Salmonella spp, as the disease progresses, the probability of detection of the organism increases. Therefore, the second fecal culture increases the sensitivity of the test to 66%. The specificity will remain the same. To reiterate, we will only perform the fecal culture on feces from horses that were negative in the first test.

  Disease + Disease - Total
Fecal Culture + 19 0 19
Fecal Culture - 10 35 45
Total 29 35 64

Utilizing the second fecal sample from the negative horses from the first sampling, we have found another 19 horses that were Salmonella positive. This produces the following table:

  Disease + Disease - Total Predicted value
Fecal Culture + 55 0 55 PV+ = 100%
Fecal Culture - 10 35 45 PV- = 77.8%
Total 65 35 100  

In this instance, we have increased our overall sensitivity to 55 (36+19)/65=84.6% from 55% and the overall specificity remains at 100%. The increased sensitivity will lead to an increased negative predictive value from 54.7% to 35/45=77.8%. This is an example of Parallel Testing.

Johne's Disease In Cattle

Parallel testing has been used in this disease as well. There is a problem with the sensitivity of the tests used for diagnosis of Johne's disease because the tests are dependent upon the stage of infection with M. paratuberculosis. Different procedures are sometimes utilized on the same sample in order to increase the detection of the organism (sensitivity).

For example, suppose we have a 1,000 cow dairy herd with a prevalence of 10% infection with M. paratuberculosis. In this example, we will use conventional fecal culture as the initial test (sensitivity=16.5%; specificity=100%) followed by the two-stage system using a two-step centrifugation technique (sensitivity=29.4%; specificity=100%). The numbers would be illustrated as follows:

  Disease + Disease - Total Predicted value
Conventional + 17 0 17 PV+ = 100%
Conventional - 83 900 983 PV- = 91.6%
Total 100 900 1,000  

We would then apply the second test (two-stage) to the samples that were negative in the first test (conventional). The sample results would be as follows:

  Disease + Disease - Total
Two-Stage + 24 0 24
Two-Stage - 59 900 959
Total 83 900 983

Using the above examples, the overall testing would be as follows:

  Disease + Disease - Total Predicted value
Overall + 41 0 41 PV+ = 100%
Overall - 59 900 959 PV- = 93.8%
Total 100 900 1,000  

Therefore, the use of parallel testing in this example increased the overall sensitivity to 41% from 16.5%, the specificity did not change and the negative predictive value increased from 91.6% to 93.8%. This is another example of the use of diagnostic testing in parallel.


  1. Michael Thrusfield, Author. Veterinary Epidemiology, 2nd Edition. Blackwell Science, Malden, MA, USA. 1995.
  2. Michael J. Murray. Disorders of the large intestine. In Large Animal Internal Medicine, 2nd Edition. Editor BP Smith, 1996. Publisher Mosby.
  3. van Duijkeren, E et al. Diagnosing salmonellosis in horses. The Veterinary Quarterly, 1995 17(2): 63 - 66.
  4. Palmer, JE and Benson, CE. Salmonella shedding in horses. Proceedings of the International Symposium on Salmonella, ED. GH Snoyenbos, New Orleans, 1984: 161 - 164.