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Police in Guatemala raided five regional offices of British aid agency Save the Children on Monday as part of an investigation into alleged child abuse.
Prosecutors requested information from US authorities in April about the alleged involvement of the organisation in smuggling children across the border, according to local media.
Save the Children said it was "aware of the activity" at their offices, and denied the prosecutors' allegations.
The prosecutor in charge of the case, Rafael Curruchiche, and the country's attorney general, Consuelo Porras, have previously been sanctioned by the United States and the European Union for attacks on democracy.
Curruchiche said the searches were being carried out in different regions of the country as part of a "transnational investigation of great importance", in a video statement on X.
The prosecutor did not name the organisation in his statement - but a spokesman for his office confirmed to AFP news agency that the offices searched were those of Save the Children.
In a statement to the BBC, Save the Children said it was collaborating with authorities but that it has "never facilitated any transfer of children or adolescents out of Guatemala".
"Following previous allegations against our organisation this year, no evidence of wrongdoing was found", the organisation added.
Earlier this year, the aid group said it was "shocked and puzzled" after its main office was raided as part of the same case.
This came just days after the secretary general of Guatemala's public ministry, Ángel Pineda, wrote a letter to Texan authorities asking for support in investigating allegations that aid groups, including Save the Children, “could be participating in child trafficking operations".
It is unclear whether Texas Attorney General Ken Paxton responded to the request.
Both Pineda and Curruchiche have previously been sanctioned by the US and the European Union, accused of trying to undermine the country's democracy. The prosecutors office denied the allegations, local media reported.
The prosecutor's office is also said to have tried to prevent President Bernardo Arévalo, who ran on an anti-corruption message and is critical of the country's judiciary, from taking office earlier this year.
Guatemala's attorney general, Consuelo Porras, was barred from entering the US in 2022 after the state department said she had "repeatedly obstructed and undermined anti-corruption investigations in Guatemala". Porras denied the claim.
Save the Children has operated in Guatemala since 1976, after an earthquake devastated the country.
It added that it "will continue working to uphold children’s rights, as we have always done, maintaining the highest standards of child protection and safeguarding in all our programs".