A 90th-minute penalty by Mateusz Klich ended Ajax’s long unbeaten run which stretched back to the end of August.
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Ajax went into the game having not tasted defeat in 20 straight matches and they started brightly with Hakim Ziyech sending a free kick just over the bar against his former side.
However, Twente came into the game and created the better chances with Enes Unal firing a shot just over the bar before Mateusz Klich saw a fierce strike saved on the second attempt by Andre Onana. Just before the break, Ajax could have taken the lead but Anwar El Ghazi decided to take a shot himself, which came to nothing, instead of squaring to an unmarked Davy Klaassen.
After the break, an anonymous Kasper Dolberg shot wide, while Nick Marsman saved a Lasse Schone free kick. Peter Bosz reacted by hooking Dolberg for Mateo Cassierra, but the change couldn’t add spark to the Ajax attack.
The game was heading for a goalless draw but in the 91st minute Nick Viergever handled in the box earning himself a red and Twente a penalty, which Klich slotted in to give the hosts the victory.
The defeat means Ajax slip three points behind Feyenoord and next up for Bosz’s side is a home tie against PSV next weekend. Twente sit 7th.