Johnson is recovering from the flu and plans to join the team ahead of Tuesday's home game against Guatemala, the last match of the semifinal round in the North and Central American region.
Castillo has a foot injury and will miss both matches, the U.S. Soccer Federation said after he had an MRI Wednesday.
Johnson has been coach Jurgen Klinsmann's first choice at left back with Castillo his backup. Because of their absences, Carlos Bocanegra and Michael Parkhurst appear to be the best options, with Geoff Cameron paired in the center with Clarence Goodson or Maurice Edu.
Jose Torres, who played left back during the opening qualifier in June, is out with a foot injury.
"Obviously it's a difficult situation losing Fabian for the Antigua game and Edgar being unavailable for both," Klinsmann said in a statement. "We were in a similar situation for the first match against Antigua and Barbuda in June, and we will be prepared. Fabian has been ill since the weekend, and we were waiting to see if he would be healthy enough to join us in Miami. At this point, it is in everyone's best interest to allow him to fully recover from his illness and come into Kansas City ready to play against Guatemala."
The U.S. squad is down to 20 players. Midfielders Landon Donovan (left knee) and Brek Shea (abdominal strain) withdrew a day earlier.
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