tag:blogger.com,1999:blog-1812431336777691886.post4058172920820325529..comments2024-02-29T03:57:00.088-05:00Comments on The Mermaid's Tale: QC and the limits of detectionAnne Buchananhttp://www.blogger.com/profile/09212151396672651221noreply@blogger.comBlogger2125tag:blogger.com,1999:blog-1812431336777691886.post-11556775502837143822015-05-25T22:01:32.523-04:002015-05-25T22:01:32.523-04:00Hi Peter, thanks for the question.
I would say ...Hi Peter, thanks for the question. <br /><br />I would say that there is always a possibility of there being false positives. The only way to know that there are false positives is to compare the method (in this case, nested PCR) to a method that is superior. In this case the “gold standard” is microscopy, but in my opinion the only reason microscopy remains the gold standard is because it has been for such a long period of time, not because it is actually a superior approach. <br /><br />There have been several studies that looked at variability in detection by microscopy, some have indicated that in low parasite density infections, that variability is greater (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504332/). What I think is going on here is that there are strong limits to detection. I would expect that a microscopist that isn’t as well trained or doesn’t have high quality lab materials would find less cases than one who is well trained, but that even one who is well trained wouldn’t be able to find as many cases as a more sensitive approach such as nested PCR. <br /><br />Our findings in this study are quite parsimonious with this explanation. Furthermore, the nested PCR was done in two rounds – the first looking for malaria at the genus level and then a second round looking for malaria at the species level – in this case we were always able to identify a species in the second level. This gives me further confidence to this approach. (If you’re interested, here is a link to our paper describing this approach, the ways we’ve tried to verify it, sensitivity and specificity: http://www.malariajournal.com/content/13/1/175 ). However, if we’d used an even more sensitive approach (such as quantitative PCR) we may have found even more undetected cases than we did by nested PCR, and some of those may have had such low parasitemias that we might not have been able to differentiate them at the species level using a second wave of nested PCR, genotyping etc. <br /><br />I don’t have the numbers with me right now, but a few of the people who were detected by PCR only did later have symptomatic malaria. This particular study wasn’t set up to look at that, the participants could have visited a malaria post or clinic that wasn’t a part of this study and we would never have known. We would also have to do genotyping of the parasites to see if the parasite strain being carried in the participant at an earlier time point was in fact the same as the parasite strain that eventually lead to that patient being ill. It is possible that a person has an asymptomatic infection (perhaps of a given genotype), is later infected by a new, unfamiliar parasite strain and then develops a symptomatic infection because of that new strain. There would be some difficulties even with this because of mixed strain infections. If we had done this though, I wouldn’t take it as a gold standard. Asymptomatic malaria is a well-known, though perhaps not very well understood, phenomenon. <br /><br />So perhaps this isn’t a very convincing answer. I think that there is a possibility of false positives, but I don’t think it would change our results here. I think that the most likely scenario is that asymptomatic, low parasitemia cases are missed because they are difficult to detect using methods (like microscopy) that are less sensitive than DNA based methods - and there are a growing number of other studies that are coming to the same conclusions. As with several other “gold standards” in tropical infectious diseases (probably in other diseases too) I think that our gold standard for malaria just isn’t very good. <br />Daniel<br />Anonymoushttps://www.blogger.com/profile/05068601494828074316noreply@blogger.comtag:blogger.com,1999:blog-1812431336777691886.post-22912243577217820502015-05-25T11:01:07.611-04:002015-05-25T11:01:07.611-04:00How are you guarding against false positives in th...How are you guarding against false positives in the PCR reaction? Did (any/some/all) of the individuals with PCR-detected malaria subsequently progress to having microscopically detectable disease?Peterhttps://www.blogger.com/profile/12559721137290332762noreply@blogger.com