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Eight militants on four motorcycles opened fire on officers who had stopped for food and then shot at a convoy protecting the health workers, police inspector general Feroz Shah said.
Speaking at a news conference, Ad Khawaja, the police chief for Sindh Province, said authorities had identified the group responsible for the twin attacks.
Pakistani Prime Minister Muhammad Nawaz Sharif released a statement condemning the attacks.
"(The officers) sacrificed their today for securing the future of our coming generations," he said.
"The entire nation will fight against these cowards with unity and unshattered resolve."
Meanwhile, Ehsanullah Ehsan, a spokesman for a splinter group of the Pakistani Taliban, Tehreek-e-Taliban Pakistan Jamaatul Ahrar (TTP-JA), claimed responsibility for the attack.
"Our attacks against security agencies and officials will continue and the Karachi attack was a continuation of this struggle to implement Islamic government in Pakistan," he said in a statement.
Previous attacks
Militants have targeted anti-polio campaigns in Pakistan for decades in an effort to beat back government influence, CNN's Nic Robertson said following that attack. Pakistan remains one of two countries -- the other one is Afghanistan -- where polio is endemic, according to the World Health Organization.
The revelation of the CIA scheme reinforced the militants' existing fear of vaccinations.