It depends - did you add the girls to an existing .girlsx file, or did you just drop a new file into the characters directory? If you did the former, you need to start a new game to get the new girls. If you did the latter, and the loader is working the way it is supposed to (I don't know that this is the case - a while back it was broken and I don't recall if it was fixed), then the game should find the new file and load the girls from it.
Put another way, the game is supposed to load new .girlsx files every time you do a save-quit-reload, but it does not load new girls in preexisting .girlsx files.
