High molecular weight (HMW) typing of Clostridium difficile is a MALDI-TOF MS based subtyping method that enables rapid and low cost surveillance of C. difficile isolates.
In summary, HMW typing is a two-step method. First, an HMW profile is determined based on the proteins detected in the 30 to 50 kDa region (figure 1). This step is performed using the direct transfer method and a special matrix solution (Ferulic acid matrix) that is able to ionize proteins of masses above 20 kDa. Most laboratories only perform step one as it is least time consuming and enables a good enough discrimination, step two is commonly only performed if an aggregation of a certain HMW profile is observed and further discrimination is desired.
For further discrimination within a subset of HMW profiles an optional second step can be performed. This second step, determines the HMW type of a sample by analysing on the presence or absence of certain biomarkers in the 2 to 20 kDa region (Figure 2).
For the best results it is recommend to analyse the isolates in the second step after performing the standard ethanol/formic acid extraction procedure. The list of PCR ribotypes, with international nomenclature analysed by HMW typing, can be found here. A full description of the method is published in PLoS ONE1 .
HMW types are directly correlated to one or more PCR ribotypes. For example HMW type 24 is a unique
HMW type that is only matched by PCR ribotype 027 *.
HMW type 14b on the other hand could be both PCR ribotype 011 or 103. To see all HMW types and PCR ribotypes read the conversion table.
Some PCR ribotypes like 011 have more than one HMW type, adding this information to PCR ribotyping data can add further typing depth in outbreak investigations.
On this site you will find a Standard Operating Protocol for performing HMW typing manually and with CDtyper, as well as instructions for the installation package on your Bruker MALDI instrument
*All 600+ PCR ribotypes have yet not been analysed with the HMW typing method. As new types are studied they will be added to the software and conversion tables. Thus, although some HMW types are to date only represented by one PCR ribotype, this can change in the future.
1Rizzardi K, Åkerlund T (2015) High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of;Clostridium difficile. PLoS ONE 10(4): e0122457. doi:10.1371/journal.pone.0122457
The CDtyper software provides you with an automatic installation package containing all files needed for performing the HMW typing method on your Bruker instrument. Furthermore, it also allows you to generate run files and upload raw data for automatic analysis of the HMW profiles and HMW types of your isolates.
Although this can all be performed manually, by using the CDtyper software you will save time both while generating run files and when analysing your results. Furthermore using a software to analyse your results eliminates subjectivity in the peak determination.
To start analysing your samples, read the Standard Operating Protocol and register for a free 6 month trial period. This website and the HMW type database are curated by the Public Health Agency of Sweden (Folkhälsomyndigheten).