A General Analysis of An In-Depth Analysis of a Piece of….Erm…

A general analysis of….An In-Depth Analysis of a Piece of Shit: Distribution of Schistosoma mansoni and Hookwork Eggs in Human Stool.

While the game of patience, CV-rewrites, recruitment calls and casual “I’m a solid team-player” line-drops continue – I’ve had a chance to read this bad-boy, along with Animal Farm and a layered spy novel by Ian McEwan (thoroughly enjoyable!).

A Bit of Background

My analysis of one of the most socially networked open access articles (from PLoS) is fairly vague (no false advertisement here), but the paper made me laugh and the title, like it did me, probably had most people in disbelief on first glance. Now, Open Access Journals (OAJs) often come under fire for their quality, by which I mean the scientific rigour of the research within a publication and to what level the submissions are actually screened, known as the peer review process. Indeed, only a few days ago we have witnessed the results of a brilliantly crafted Sting Operation which highlighted the desperately worrying trend of novel OAJs appearing with fictional editors and fabricated institutions along with miserable peer review performances. I mean – truly dismal performances. Well done John Bohannon! Despite this, PLoS faired well in the Sting Operation despite more general concerns from academics over the quality of scientific papers that PLoS typically publish. All the more reason to give this ‘In depth analysis of a piece of …….’ paper a read and see personally whether its’ conclusions have any grounding.

A hilarious title is not completely novel in academic research – take this one about coked up honeybees for example, or any on this list compiled by WIRED magazine. Either way, the concept of this actual study is well within reason and there are some interesting and important implications to come out of the sound findings, such as how Doctors determine the diagnosis of a helminth (parasitic worms) infection and potentially how future studies, which determine the effect of medicines designed to act against these worms (antihelmintic drugs), are designed.

I made a friend in Vietnam who wanted to focus her PhD on intestinal worms. She had this cool hypothesis (that was still a growing idea) about immune system and worm ecology, which made me scratch my head for struggling to understand better, but she never told me just how big of a worldwide issue they pose. The WHO reckons that about 24% of the World’s human population is infected with a soil-transmitted helminth. Now this doesn’t have to mean it’s an issue – but it can be. Especially if you’ve been infected with a relatively big load of them, which can lead to diarrhoea, stomach pains, neural impairments and blood loss. Epidemiological surveys, which typically investigate stool samples for evidence of parasitic worm eggs, show the highest burden lies in the tropics and sub-tropics. However despite knowing this and having drugs available to control helminth infections, public health researchers and clinicians commonly share the concern that helminth infections represent, with immensity, a neglected tropical disease.

Hence the study holds two ultimate aims, 1) to determine whether the eggs of helminths Schistosoma mansoni and Hookworms exhibit any particular spatial distribution in the piece of, erm…ahem….faecal matter… and 2) to understand what might constitute a more accurate diagnosis. Sounds like a perfectly reasonable endeavour for the advancement of medical knowledge for such a high burden disease right? Yes (say yes).

The Study’s Execution

222 individuals from Ivory Coast were invited to participate in this study. For the ‘field collection’, the instructions as presented in the ‘INSTRUCTIONS’ figure below, were given to all participants. Notice that the timing is recorded, which is particularly important as the time delay from the stool production appears to influence diagnostic sensitivity – especially for hookworm.

There is such a thing as the Bristol Stool Chart (BSC; nice one Bristol!) which is a medical aid to distinguish human stools into categories. Here, the authors place specimens into 5 distinct categories:

1) Sausage-shaped (equivalent to type 3 on the BSC)

2) Sausage-shaped but lumpy (type 2 BSC)

3) Sausage-shaped but soft (type 3 BSC)

4) Lumpy (Type 1 BSC)

5) Mushy (Types 5-7 BSC).

…and then (randomly) either underwent whole-stool homogenisation (high frequency mushing, often performed with tiny silicon beads) or underwent direct helminth egg spatial disbtribution examination by drawing lots. Regardless, fecal egg counts (FECs) were taken before and after homogenization.

The authors are very thorough in explaining what they did, giving details of how deep the cut into the faecal samples and the tools they used throughout. They also give a solid account for time as a confounding factor and therefore, randomly allocate the times of stool sample analysis, with each examination time point lasting 2 hours. No qualms here – brilliant stuff. However, what exactly are the four distinctive examination time points that these researchers claimed to have established? Why not mention what they are at least once throughout the paper? Wouldn’t future researchers / diagnostic teams want to know???? Bizarre. Also, the researchers follow WHO guidelines for the Kato-Katz ‘thick smear’ preparation and examination which is common practice, despite the authors indicating shortcomings of this method from the start-off (see below).

Regardless of the processing schemes, an approximate third of all samples were stored in the following manners to determine their effect on egg degradation, with the obvious aim to determine how best to store stool samples in the future for accurate helminth diagnosis:

1) Stored in a box on ice

2) Stored covered with a tissue soaked with tap water

3) Placed in shade outside of the laboratory without any additional preservation effects.

Problem…

We are never convincingly told exactly why the above storage methods were chosen. I suppose we are meant to assume that these are typical storage procedures during helminth diagnosis. However, because of relative uncertainty of how best to go about storing these stool samples, it makes for interesting commentary in the results.

Incredible Details

Instructions

Instructions

This is how to do it – Field Workers: Watch and Learn! This is what I want to see in every set of instructions I see from now on. Something I can relate to. Direct. Clear. Scientifically astute. Makes me laugh.

Sausage and the Kato-Katz

Sausage and the Kato-Katz

So, prior to homogenisation, the researchers chopped the ‘sausage shaped’ faecal samples into four pieces and performed the Kato-Katz procedure on central and surface areas, in preparation for the search of parasitic worm eggs.

Problem…

The investigators are using the Kato-Katz method to process the stool samples in preparation for the search of eggs. This is still a common procedure however, even the authors state problems of reliability in the detection for parasitic eggs in faecal samples. So if the intent of study is to get to the bottom of helminth distribution in faeces, why not go one step further by utilising other tests to strengthen the validity of results, such as this.

Shit Results

Not really! They’re quite cool actually. Of the 222 participants, 125 were positive for helminth infection, but only 116 had enough stool for analysis (52.3%). There’s an approximate third split for the storage method, followed by categorising the samples into ‘suitability of assessment’ for

- egg location along the length axis (n = 45)

- egg location along a cross-sectional axis (n = 35)

And then, OF THESE, also…..

- homogenisation of stool parts (n = 62)

Note: 53 ‘whole stools’ (i.e. they are not cut up into tidy pieces) undergo direct homogenisation.

By then performing Kato-Katz thick smear preparation and microscopy examination, the investigators identified five different helminth infections under varying prevalences and infection intensities. Amazingly, 5.6% of infected samples showed evidence for TRIPLE worm infection, with 20% showing DOUBLE worm infection. They sure do get about…. It’s important to note that for the rest of the statistical anaylses, the investigators only ever really focus on S. mansoni and Hookworm.

SPATIAL DISTRIBUTION: No clear pattern was observed although in hookworm infected individuals, higher egg counts were significantly noticeable in the front-ermost piece of stool sample than the back-ermost piece.

TIME and STORAGE in EGG DECAY:

- For Schistosoma mansoni the egg count results did not differ between the different time points regardless of how they were stored.

- For Hookworm positive samples, it’s a different story. The egg counts do decay over time UNLESS if samples were stored on ice or ‘kept humid’ they say – which I assume means ‘covered with water soaked tissue’???

Problem…

No reporting of a blinded assessment of outcome. This is a shame really, considering the investigators were on a good roll (concerning laboratory practice) by stating randomised allocation of the 5 different categories of stool samples into either full stool homogenization or ‘piece’ homogenization.

Even though the statistical analyses back up their findings, we’re only dealing with one immediate population from Ivory Coast here. However, it has to start somewhere and this is a step in the right direction towards a deeper, more universal understanding of helminth diagnosis and how to conduct parasitic worm epidemiological surveys.

5 Point Conclusion

- Solid piece of necessary work with the aim of improving diagnostic procedures for this high burden, neglected tropical disease.

- Hookworm eggs exhibit time-dependant decay, unlike Schistosoma mansoni eggs. This can be avoided if samples are kept on ice. However, I would still love to know what the ’4 distinctive time points’ were that they established – surely this is useful and necessary diagnostic information? Either way, a good general rule of thumb to take away from this would be to store stool samples on ice (if possible, within the sweltering heat of the tropics) and don’t dither about the examination process for accurate diagnosis. Both of the aims of the study I set out at the beginning of this piece, are generally well met.

- Why question the Kato-Katz method and then stick with it without adding more for validity of testing?? A few laboratory procedures could be improved – such as blinded (or double blinded) assessment of outcome, which in this case is faecal egg count.

- The direct results are only relevant to Ivory Coast. However, it paves the way for more in-depth analyses  throughout the rest of the tropics / sub-tropics and does provide useful, novel and researched information for the conduction of future surveillance studies.

And perhaps of most interest…..

- The Title is well chosen. It made you read it. It made this article one of the most widely circulated Open Access papers among social networks. The authors even poked some light-hearted fun at it in their conclusion by quotation marking piece of shit as if to say….”yeah, that’s right – we did that!” Great stuff, and it brought a smile to my face. Schistosomiasis and helminth infections in general really are what we define a ‘neglected tropical disease’ and given that many top-level researchers devote their time to addressing this, I can fully understand their desire to share their findings in the hope it inspires more people to rally against this burden of momentous proportions. Good on ‘em.

Thank you for reading my fairly vague analysis on an in-depth analysis of a piece of youknowwhat.

Phil

______________________________

Next time on Philandtropical:

How Will Smith turned from this…… 

Fresh Prince

Fresh Prince

…. to this…..

Will Smith Ali

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