Molecular tests for the detection of up to 37 subtypes of the Human Papillomavirus (Papillomavirus or HPV)

When taking the swab, it is recommended to store it without any transport media. If PBS is added (maybe because other exams are made that require this PBS), then the sample should be stored frozen until the extraction. Otherwise, other microorganisms might grow, increasing DNA quantity and degrading the sample. Before the extraction, the dry swab is then resuspended in 500 µl of PBS in a 1,5 ml tube, mixing with vortex for 1 minute. After that, centrifugue the tube for 2 minutes, at 18.000 g. After discarding the supernatant, add 500 µl of PBS, and resuspend the pellet by vortex. Centrifugue the tube for 2 minutes, at 18.000 g. Discard the supernatant. Resuspend the pellet with 200 µl of PBS, and proceed as indicated in the protocol (20 µl of proteinase K + 20 µl buffer AL, etc). By doing that, interferent material will be eliminated, and the PCR will be “cleaner”.

Generally, it is not possible to calculate a diagnostic sensitivity and specificity, but rather a concordance of sensitivity and specificity between 2 techniques, as neither is really the Gold Standard. In other words, only how one compares with the other can be said. On the other hand, the techniques compared do not detect exactly the same types. This means that, if one of the types not detected by one of the techniques is present, there will be a discordance. Not because of a false result, but because the test is not set up to detect it.

One possible explanation is that the patient becomes infected and recovers between tests. If its a mild infection and the immune system is strong, the patient may overcome the infection and be re-infected with other types by the next test. There is also the possibility that the patient may have co-infections with several HPV types, but that all of them have a very low viral load. In these cases, it is possible that not all types will be amplified in each test, but that they will be amplified randomly. Since the types are not repeated, this does not seem likely, but cannot be ruled out. Another reason could be that there were contaminations in the different assays. It is unlikely to have occurred in several assays in a different way, but this should always be checked by placing blanks in all assays to ensure the veracity of the results.

Possibly contamination. Blanks should be tested: if they are correct, then the result is valid. This is not surprising because multiple infection in these cases is common. On the other hand, if the blank test comes back contaminated, then there is contamination. In this case, the reagents should be discarded and the cleaning protocol should be performed. No further samples should be tested until several valid blanks are obtained.

It may be a type not present in the kit or at a low concentration.