Britain’s Heather Watson is among the 47 players who had already been told they had to stay in their rooms for 14 days following positive tests for coronavirus on flights that had taken them to the country.
And a similar situation has occurred involving a flight arriving from Doha, with one positive case leading to isolation for 25 players.
A statement on the official Twitter account of the Australian Open said: “One positive Covid-19 test has been returned from a passenger on a charter flight into Melbourne from Doha which arrived at 5.30am on January 16.
“The passenger is not a member of the playing contingent and had tested negative before the flight. There were 58 passengers on the flight, including 25 players. All are already in quarantine hotels.
“The 25 players on the flight will not be able to leave their hotel rooms for 14 days and until they are medically cleared. They will not be eligible to practice.”
A fourth positive case has been recorded from the two flights that carried the initial group of 47 players.
Covid-19 Quarantine Victoria commissioner Emma Cassar revealed the news on Sunday, adding the positive test was returned by a broadcast team member on a charter flight from Los Angeles.
Two other people on that flight had earlier tested positive, while another case was detected on a flight from Abu Dhabi.
Cassar said: “It’s important to note that all the four positives all had a negative test result prior to boarding the plane.”
That was after Australian Open tournament director Craig Tiley insisted players were made aware of the risks of being put into isolation on arrival in the country.
Cassar said some “low level” quarantine breaches among players had been reported, including “a player who opened his door to try and have a conversation with his training mate down the hallway”.
She added: “He’s got a phone, you can pick up the phone and use the telephone as opposed to putting you and others at risk. It is really low level but really dangerous acts which we just can’t tolerate.”
Tiley and his team worked for months to secure an agreement whereby players would be allowed out of their rooms for five hours each day during the two-week quarantine in order to practice and do gym work but those carefully worked plans are now in tatters.
A number of players reacted to the news with shock and anger on social media, and Tiley was sympathetic to those feelings.
He told Channel 9 TV: “I totally understand the emotion the players are going through right now as well as their objection to the situation.
“The determination of who was and who wasn’t a close contact was going to be entirely up to the health department, and they’re doing what they deem is necessary in order to keep our community safe.
“Obviously what has changed over the last several weeks is the new UK strain, which is more infectious, and there’s obviously a great desire by all of us to make sure that doesn’t come into our community.
“These conditions, they are constantly changing, but there’s always a risk. We did make it very clear at the beginning, that’s why we had the player groups in cohorts, there was always a risk that someone would be positive then and have to go into 14 days of isolation, there was a risk on the plane that you would be a close contact, there was a risk that everyone could be a close contact.
“It is unfortunate that we’re in an environment now where we’ve got to manage it.”
British hopeful Watson is among the 47, along with grand slam champions Victoria Azarenka, Angelique Kerber and Bianca Andreescu, whose coach Sylvain Bruneau revealed he was the source of one of the positive tests.
The players should be out of isolation before the week of warm-up events begins on January 31 but they will clearly not be in peak condition to compete.
Tiley dismissed any suggestion the Australian Open, which has already been pushed back by three weeks, could be delayed further but said the players will be supported in any way possible, including with the delivery of exercise equipment to their rooms.
Additional reporting by PA.