The family of missing Ben Needham, from Sheffield, say they remain "strong and resolute" as they continue their search for him after a DNA test on a young man on Cyprus proved negative.
Ben's mother, Kerry Needham, and her family spent Ben's 24th birthday waiting for results of tests on a young man featured in video footage shot in Cyprus that emerged this week.
But last night the official Help Find Ben Needham Facebook page confirmed the DNA comparison analysis was not a match.
The post said: "The Needham family have, in the last few minutes......had confirmation that the DNA results on the man in Cyprus have came back as negative.
"This, again, is more disappointment for Kerry and her family. However, they remain strong and resolute in their search for Ben, and there are other leads currently being looked into which keeps their hopes alive."
Ben went missing on the Greek island of Kos 22 years ago, when he was a toddler.
The man who handed himself in to authorities in Cyprus this week has facial similarities with reconstructions of how Ben may now look, the family said.
He came forward after a video emerged of him at a church in Limassol, on the island.
Ben vanished on July 24, 1991 after travelling to Kos with his mother and grandparents, Eddie and Christine, who were renovating a farmhouse in the village of Iraklise.
Over the years there have been a number of possible sightings and a range of theories about what happened to him.
Last year a major new police operation focused on a mound of earth and rubble close to where Ben was last seen but no trace of him was found.
Police in Cyprus have said that DNA tests on a man who could have been missing Ben Needham have come back negative.
Ben's DNA had been released to police on the island after it was thought the man could be the boy who has been missing since 1991.
South Yorkshire Police have released the DNA of Ben Needham to the Cypriot authorities at their request.
Greek authorities are still investigating Ben's disappearance in island of Kos in 1991.
The family of the missing Sheffield toddler Ben Needham have suffered another set back after it emerged that a young man being investigated in Cyprus is probably not Ben.
The man, said to bear a resemblance to one of the updated e-fit pictures which have been released in the search for Ben, handed himself into the Greek police this afternoon and has undergone a DNA test voluntarily.
The results of the DNA test should be known in two or three days. But tonight Kerry Needham played down the development saying the Roma family he was with were related to his girlfriend.
Police in Larissa have confirmed to Calendar that they have received calls about Ben Needham,among the hundreds of inquiries about Maria, the girl who was recently discovered in a gypsy camp in northern Greece.
Although South Yorkshire Police are ruling out any connection with the Sheffield toddler, who disappeared from Kos in 1991, Ben's mum Kerry says the case proves children can **be hidden from authorities.
Kerry is calling for detectives to further investigate the camp to see if anyone in there has been involved in child trafficking.
An image has been released of a Roma couple with the young girl, known as Maria, who they are accused of abducting as a baby.
The couple have today been appearing in court in Larissa, Greece.
Kerry Needham, mother of Ben who has been missing since 1992, has been speaking to Calendar's Claire Ashforth about the renewed hope of finding her son after the discovery of a girl in Greece.
A couple are presenting their case in court after a young girl was discovered at a Roma camp in Greece.
A dental examination has revealed the girl, known as "Maria", is aged between five and six years old.
ITV News reporter Lewis Vaughan Jones is outside the court in Larissa, where the couple are giving evidence:
The Greek charity taking care of the girl known as Maria said the result of a dental examination shows she is between five and six years old.
The leader of the local Roma community where four-year-old blonde girl, known as Maria, was found said children are have not been bought and sold in his town.
Community president Babis Dimitriou said: "No such thing has happened in our community.
"Is the woman who found the child at fault? She should have gone to the police [and say] 'the Bulgarian abandoned the child with me and got up and left.'"