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Two health workers exposed to a patient with the Middle East Respiratory Syndrome (Mers0) have begun showing flu-like symptoms.
One has been hospitalised while the second is being monitored at home, officials at the Dr. P. Phillips Hospital in Orlando, Florida said.
Eighteen more people are now being tested for the deadly disease, which is not thought to be a threat to the general public.
President Barack Obama has been briefed on the two confirmed cases.
A statement from the White House said: "The CDC [US Centers for Disease Control and Prevention] is taking the current situation very seriously."
As the first US case of the MERS virus has been confirmed in a man who flew from Saudi Arabia to Chicago via the UK, health officials sought to advise Britons who may have traveled on the same flight as the affected passenger.
The man, a healthcare worker, traveled via a British Airways flight on April 24 from Riyadh to London, where he changed flights at Heathrow airport to fly to the US.
He landed in Chicago and took a bus to Indiana, before experiencing respiratory symptoms a few days later.
Health officials are in the process of contacting British airline passengers who may have made contact with a passenger who has been hospitalised in the US with the often fatal MERS (Middle East respiratory syndrome).
The man, a non-British national, took a British Airways flight 262 on April 24 from Riyadh, the capital city of Saudi Arabia, to London, where he changed flights at Heathrow to fly to the US.
Public Health England said it has contacted UK passengers on the BA flight to London who were sitting in the vicinity of the affected passenger and is working with US health authorities to contact any British passengers on the April 24 onward flight to Chicago, American Airlines Flight 99.
It said that the risk of the infection being passed to other passengers "is extremely low" but was contacting the passengers "as a precautionary measure."
Any UK based passengers on Flight 262 on April 24 who have since become unwell or experienced respiratory symptoms, such as shortness of breath, are advised to contact NHS 111.
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While the risk of infection is very low, the Mers virus kills around a third of people infected and has no cure.