Article: Simultaneous Detection of Entemoeba, Giardia, Cryptosporidium

Question 1

Why performing PCR/quantitive method instead of microscopy for Entemoeba hystolytica?

Answer 1

1) You cannot differentiate between E. histolytica or E.dispar under microscope, and E.dispar is a more common noninvasive entemoeba
2) Many infections are also missed because sensitivity of microscopy is low
3) Entemoeba can cause colitis dystentery and liver abscess, causing 100,000 deaths a year so its really important to diagnose it properly

Question 2

Why performing G.lambria PCR?

Answer 2

Actually, microscope and ELISA is used, but PCR also has amazing specificity and sensitivity compared to microscopy and ELISA
It’s a very common disease, one of the main nonviral cause of diarrhea in industrial countries - so if its that common its important to diagnose it in a public health perspective

Question 3

Why performing C.parvum PCR?

Answer 3

1) Microscopy requires acid-fast staining, its more laborious
2) Although staining is performed, sensitivity and specificity of microscopy is rather low because identification really depends on the skills of the technician
3) Antigen ELISA: found to have low sensitivity and showed crossreactivity with other species
4) Also PCR was shown to be decent in fecal samples
5) C.parvum normally is also very common: but its also very critical for AIDS patients so it might be also life-threatening in immunocompromised - important to have this sensitivity and specificity

Question 4

Why use all three species together for a PCR? (pros and cons)

Answer 4

1) Usually all these diseases look like each other: they are diarrheal diseases so clinicians often question all three of them during diagnosis
Therefore why not do all of them together and verify them
2) In all those diseases PCR is either shown to be useful, or preexisting methods are shown to be not sensitive/specific enough
3) Also no one really uses separate PCRs for separate diseases, its not really handy to do that, running separate PCRs for each suspicion
So its good to couple them all together: pushes people to use PCR more
4) Now you have less lab cost, less labor time, less contamination with fluorescent detection probes in RT-PCR - also they added internal control to see any specificity issues

-Cons:
DNA extraction from fecal sample was hard apparently, now improved also

Question 5

Results?

Answer 5

-The assay was specific
-The assay did not get affected by the presence of other DNA in samples, or multiple primers = so the performance of multiplex PCR was as good as regular PCR
-Also apparently once there are more species in feces, often PCR gets inhibited/or amplifies other things: they put samples from:
1) people that they don’t know whether they are infected or not
2) people w E. dispar
Both showed no inhibition or nonspecific amplification
-Detection rate was %100 (%100 specificity): each species was detected in around the same cycles (cycle threshold in each species was similar)

Question 6

Which sample is used for detection?

Answer 6

Feces

Question 7

Conclusion

Answer 7

-They concluded that for each species: PCR was more sensitive than microscopy at least, also provided early detection: one immunocompromised kid showed C.parvum earlier w PCR compared to microscopy
-They thought you could expand this: for eg. you can make E. histolytica + Giardia + another species: and apply this on the travellers from tropics
2 more species + C.parvum: for immunocompromised
-This is great for parasitology diagnosis labs/can it be applied for settings like Africa? needs further studying