Monday, August 31, 2015

BtB#3- Epidemiological typing


Often, we talk about epidemics, infection outbreaks etc. The technology has advanced sufficient enough in past 10 years for us to efficiently track and come up with epidemiological pattern in a matter of days. Organisms are seldom talked in terms of species. Rather we prefer, to talk in terms of strains, sub-types, genotypes etc. With the ability to sequence an entire bacterial genome in less than a day, molecular epidemiology is a simple thing (Not really!! But compared to what was something a decade ago, it really is simple). Often you would read publications on genotypic epidemiology with fancy graphs and pictures. But how many of us have given thought on how to type? Basic question.

Fig 1: Typing methods.
For beginner's, lets talk first about a little basics here. The earliest of typing methods involve techniques such as differentiating based on ability to perform a chemical reaction, ability to grow in presence or absence of some important nutrient, ability to be attacked by a particular set of phages etc. It had a low resolving power. It cannot be used to differentiate a very close strain. With improvement in techniques, it was recognized that there are a variety of antigens which vary and hence used for typing. This was a classic method for a longtime. For example, E coli O:157 H7. These are phenotypes, which means based on phenotypic expression. With improvements in genetics, genotyping was the technique of choice. You get an idea of the genetic code. Restriction pattern was one of the classic genotyping methods. The idea is that if the restriction enzyme cleaves at a particular site in the DNA which changes with strain you can get a pattern, discriminating enough. This technique is known as RFLP (Restriction Fragment Length Polymorphism). And then came the sequencing. From sequencing a single gene (Uni-locus typing) to sequencing several genes (Multi-locus typing) several mutations could be captured. More recently this has been expanded to include Whole Genome sequencing.

Fig 2: Criteria
But there is a large array of possible combinations in all the methods. Let's take a case. How did one come to decide that O and H are the antigens that needs to be typed? There are 4 important criteria that determines the use of typing method- Typeability, Reproducibility, Discriminatory power, ease of performance and interpretation. Here are a couple of considerations. I will use O-antigen as an example. But the rule extends to any method and its target.

1. The target which is to be typed  is sufficiently variable. That means, if all the E coli is expressing the same antigenic "O", then it is not of potential target.

Fig 3: Delicate balance between variability
and hyper-variability
2. The target is not hyper-variable. If O antigen is varying  in every 10 generations, then by the time the culture is obtained, it has changed. Also it is too much to keep track of. In other words, the clones of the strain should be of same type and stable

3. If there are 2 targets it should be resolvable. That means there are no confusing reactions. in other words technique should be able to discriminate between two different types even when they appear to superficially related.

There are a set of strains called as "Untypable strains". There will occasionally be a strain that cannot be typed by one method. Such strains are called as untypable. For example, there are Spa-untypable S auerus strains. But they can be typed by other genetic methods.

That means you have to select a target that has sufficient chance to vary but doesn't vary so often (Or lets simply call it stable). With genetic methods, there is an additional advantage of estimating evolutionary pattern and hence we can cluster the types and follow lineages. There is a very detailed discussion about the epidemiological typing methods in Microrao and recommend you have a look at it (Link).

One  more question. What is the point of typing. Isn't it enough to know the pathogen.

There are several answers to this question. The first important point is, different strains have different pathogenic ability despite being the same species. For example, USA 300 which is a particular clone type in S aurues has higher pathogenic ability than some of its other counterparts. 0157 H7 strains of E coli are known to be notoriously pathogenic causing complications in comparison to other types. The second point is tracking. When there is a sudden increase in number of a particular case typing helps to track down the source of infection and also the pattern in which it is moving. By knowing these parameters it is possible to control these issue and thus reduce the spread of such an infection.
van Belkum A, Tassios PT, Dijkshoorn L, Haeggman S, Cookson B, Fry NK, Fussing V, Green J, Feil E, Gerner-Smidt P, Brisse S, Struelens M, & European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group on Epidemiological Markers (ESGEM) (2007). Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clinical microbiology and infection, 13 Suppl 3, 1-46 PMID: 17716294

Struelens MJ (1996). Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clinical microbiology and infection, 2 (1), 2-11 PMID: 11866804

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