The finding supports the WHO's belief that limited human-to-human spread occurred in the family but that transmission stopped in late November, a WHO influenza expert said in an interview from Geneva.
Dr. Frederick Hayden said confirmatory tests on specimens taken from a number of other members of the family were not positive. But that shouldn't be taken as a sign that they were never infected, he warned.
"I think that there's a probability that other individuals in this family were in fact H5N1-infected," said Hayden, adding that checking the blood of surviving members of the family for antibodies to the virus should reveal how many infections actually occurred in this cluster.
"Until the followup serology (blood testing) is done, we can't be strong in saying that H5 has been ruled out in any of these people. I think especially in the family unit one would want to be cautious," he said.
"But the good news, of course, is the epidemiologic data are secure that this is not an ongoing event."
The news comes at a time when H5N1 activity is on the upswing. In recent days human cases have been reported by Indonesia, Vietnam and Egypt.
The confirmed positive test came from a man who was the third member of a large family to become sick after one brother, a veterinary worker, was involved in culling poultry infected with H5N1. The man who tested positive died on Nov. 28.
Testing done by Pakistan's National Institute of Health earlier in December identified nine possible cases, five in the family cluster, a female doctor who had treated members of the family and three poultry cullers who were unrelated to the family. A 10th possible case, another brother in the family, died without having been tested.
Some of the testing was done using a PCR or polymerase chain reaction test that looks for traces of virus in sputum samples. In other cases the Pakistan lab looked for antibodies in blood using a test which has not been validated as effective in finding H5N1 infections.
Retesting by the WHO collaborating centre for influenza in London and by experts from a U.S. naval laboratory in Cairo - known as NAMRU-3 - did not support the initial positive findings on all the suspect cases, Hayden admitted.
The negative results may be the product of degradation of specimens due to multiple freezing and thawing of the samples, he said. Other factors could have affected the results, including when in the course of infection the specimens were taken or whether suspect cases were started on antiviral drugs before a sample was taken.
As well, tests on some of the suspect cases point to infection with human influenza viruses, not the avian strain. Hayden said it appears the Pakistan laboratory may have set a low sensitivity threshold in their testing in order to be sure to catch all possible cases, but in the process found some human flu infections as well.
Testing on the doctor and the three poultry cullers, who all survived, suggests none was infected with H5N1, though one of the cullers tested positive for the human flu strain H1N1.
The doctor, who had a cough and what looked like a head cold, tested negative in London and by the NAMRU experts.
"To me the available evidence would suggest that she did not have an H5 illness," said Hayden, adding blood testing of the doctor and other health-care workers who cared for the brothers would be important.
The strongest evidence of infection appears to be among four of the brothers. Hayden said for these men, there was either X-ray evidence of pneumonia or pneumonia was suspected because of their symptoms.
The cluster dates back to late October, when the veterinary worker who was involved in culling poultry infected with H5N1 fell ill. The man, who required hospitalization, survived his illness.
Two brothers who cared for him, both before he entered hospital and while he was there, later fell ill and died. Hayden said investigations in Pakistan suggest neither of these men were exposed to infected poultry, pointing to the veterinary worker as their source of exposure.
One of the brothers died on Nov. 19; he was not tested for H5N1 and cannot be added to the list of confirmed cases.
The other died on Nov. 28. (The WHO had earlier said the man died on Nov. 29.) This man was the first in the cluster to be tested for H5N1 and his positive test was the first reported by Pakistan. It was this specimen that also tested positive in London and by scientists from NAMRU.
A fourth brother was also hospitalized for illness around the same time. He survived.
Two more brothers who lived in Pakistan showed no symptoms of illness but were tested. Initial testing suggested one was positive, but that was not confirmed by the London lab, Hayden said. He noted the testing by the NAMRU experts suggest the man may have had influenza, but not H5N1.
"He's another one where I think followup serologic testing is going to be very important," he said.
Yet another brother, who lives on Long Island, N.Y., travelled to Pakistan with his son to attend at least one of the funerals. On their return to the United States they suffered mild respiratory symptoms and consulted their family doctor, who alerted New York State public health authorities.
In early December, the U.S. Centers for Disease Control sent a plane to Albany, N.Y., to collect samples taken from the two. Testing at the CDC's labs in Atlanta confirmed the man and his son were not infected with the virus.
A cousin who lived near the family and who worked at the hospital where the brothers were treated, was also ill with fever at the time. He initially tested positive for H5N1 but testing by NAMRU and in London suggests he was not infected with the virus.
To date there have been 343 confirmed H5N1 cases in 14 countries, with the virus claiming the lives of 212 of those people.
