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Kolarov played all 90 minutes as his country succumbed to a late 3-2 defeat to their already-eliminated hosts, knowing that a win would have booked their ticket to the tournament in Russia.
Serbia must now beat Georgia next week to guarantee their qualification.
Elsewhere, Hector Moreno also played the full 90 minutes as Mexico continued their good form with a 3-1 win over Trinidad & Tobago.
Hirving Lozano, Javier Hernández and Héctor Herrera all scored late on to turn the game around for El Tri, who are already booked in for next summer's tournament.
In Europe, neither Stephan El Shaarawy (slight injury) or Cengiz Under (on the bench) played a part as their respective nations suffered disappointing nights.
Italy were held to a 1-1 home draw with Macedonia, while Turkey slumped to a 3-0 defeat against Iceland.
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