How scientists discovered false evidence on the world's "first Aids victim"
US experts have destroyed claims that the death in 1959 of a British printer was caused by HIV
The 25-year-old apprentice printer was in reasonably good general health until he developed breathlessness, night sweats, tiredness and loss of weight in December 1958. From then on, his health deteriorated radically and dramatically.
Scaly brownish lesions had appeared on his skin. He developed a fever and a painful anal ulcer appeared in February 1959, followed by another ulcer between his nostril and upper lip. "He was wasted, febrile and ill,'' his doctors wrote.
Mr Carr was admitted to the Royal Infirmary on 8 April but nothing the doctors did seemed to help him. The breathlessness and wasting became severe and he died officially of pneumonia caused by Pneumocystis carinii, an "opportunistic'' infection that takes advantage of a weakened immune system, and cytomegalic inclusion disease, a viral infection causing the cells of the lungs to become enlarged and damaged.
The case of David Carr was so unusual that the two doctors and the hospital pathologist studying him decided to describe it to the medical community by writing a detailed description in the Lancet. It was published on 29 October 1960.
Trevor Stretton, medical registrar, John Leonard, senior registrar, and George Williams, the pathologist who carried out the autopsy on Mr Carr, had no idea what was responsible for their patient's fatal fall into ill- health. Judging by the complete lack of response to their Lancet paper, neither did anyone else.
David Carr remained a medical enigma until the early 1980s when it dawned on the three doctors that their patient had symptoms similar to those beginning to appear in medical literature due to a mysterious new illness: Aids. In 1983 they wrote to the Lancet posing the question: Did our patient have Aids?
They pointed out that the man - who was not then named - had been in the Navy between 1955 and 1957 when they supposed he had travelled abroad. "He was not married and we know nothing of his sexual orientation,'' the doctors wrote. The implication was that the man could have been homosexual and might have picked up the Aids virus whilst overseas. Aids researchers took a keen interest in the case and this encouraged Dr Williams to locate the tissue samples he took in 1959 from the corpse. Although it was standard practice to store such samples, the unusual nature of death led Dr Williams to take more tissue pieces than usual.
He sliced small portions of tissues from just about every organ in Mr Carr's body. Dr Williams said he preserved more than 40 samples and embedded them in paraffin blocks.
Each set of tissues was given a unique number which could be used to cross-reference the samples with the post-mortem examination records of each case in question. Dr Williams said that there are "thousands'' of stored tissue samples in the pathology department of Manchester University. "When I was looking for the tissues for this patient, I came across blocks from patients just after the last war,'' he said.
Dr Williams said he located the tissue samples taken from Mr Carr in 1987. Unfortunately, there was no test for HIV at the time that was sophisticated enough to use in such dried-out material. (The HIV antibody test was designed to work primarily on blood serum).
Then, towards the end of the 1980s, scientists had developed a new technique for amplifying minute quantities of DNA - the genetic blueprint - from all manner of tissue fragments. The polymerase chain reaction (PCR) test had revolutionised forensic science and was now about to be employed on the mortal remains of David Carr.
Dr Williams sent some samples to Gerald Corbitt, in the hospital's virology unit. Together with his research assistant, Andrew Bailey, Dr Corbitt applied the PCR test to the tissue and had a positive result: they found that HIV had infiltrated the DNA.
Dr Corbitt, however, wanted to be absolutely sure that this was not a "false positive'' result. He was well aware that the PCR test was so sensitive that it could quite easily amplify any stray molecules of HIV that may contaminate the samples, so he asked Dr Williams to send him some more tissue, but this time in a proper "blind'' trial.
Dr Williams therefore sent Dr Corbitt 12 tissue samples in separate tubes. Six came from David Carr and six from a man of a similar age who had died in a traffic accident in the same year. Neither Dr Corbitt nor Mr Bailey knew which sample came from which patient because only Dr Williams had access to the code describing what each tube contained. Dr Williams said he kept the code in a locked drawer and no one but himself had seen it. "When I say no one, I mean no one,'' he told the Independent.
The blind experiment went ahead. Mr Bailey, who did much of the bench work, performed the PCR test on each of the 12 tissue samples. He and Dr Corbitt went to extraordinary lengths to avoid contamination because they knew how sensitive the PCR test can be.
The work was done in a laboratory where, as far as they know, researchers had never handled HIV. As an added precaution, half a dozen different rooms where used for each stage of the experiment and the scientists wore disposable gloves, gowns and hats while working with the samples in an air-filtered hood.
They had even asked Dr Williams to slice sections off the stored tissue blocks using different laboratory knives. Dr Williams said he also washed the knives in alcohol to make absolutely sure there was no cross-contamination.
Mr Bailey repeated the PCR experiment twice and got the same results each time: four of the tissues were positive for HIV, eight were negative. It was left to Dr Corbitt to phone over the results to Dr Williams, who had the code to hand.
Dr Corbitt read the results through "one by one'' and was told that the four positives all came from David Carr. Kidney, bone marrow, spleen and throat tissue all had HIV present. Tissue from Carr's brain and liver were negative, as were all the tissue samples from the "negative control''.
The results surprised Dr Corbitt because they were better than he ever expected. "An occasional false positive wouldn't come as any great surprise, so to get the correlation of the sort we got did surprise me,'' he said.
With Mr Bailey and Dr Williams, he quickly submitted the results of the research to the Lancet, which published them as a short letter on 7 July, 1990. The resulting international publicity was huge. The three researchers, along with Dr Stretton and Dr Leonard, were feted on both sides of the Atlantic. The New York Times proclaimed: ``Puzzle of sailor's death solved after 31 years: the answer is Aids.''
Dr Williams remembers well the day when the news story broke. He was on a fishing holiday in Scotland, driving across lonely moorland at about 7.45am listening to Radio 4's Today programme. He heard the late Brian Redhead talking about a young pathologist in Manchester who had performed an autopsy in 1959. "It was almost as if someone was speaking to me,'' Dr Williams recalls.
News of the "1959 sailor'' -people had not realised then that David Carr, as a national servicemen, was not a professional seaman - once again stirred intense interest in the United States. In particular, it intrigued a committee of scientists set up to investigate allegations in Rolling Stone magazine that Aids could have come about as a result of mass polio vaccination campaigns in the Belgian Congo between 1957 and 1960.
In March 1992, Rolling Stone had published an article written by the journalist Tom Curtis suggesting that the Aids virus might have been transmitted inadvertently from monkeys to humans because monkey cells were used to make the polio vaccine and it was understood that the simian immunodeficiency virus - SIV - was the most probable ancestor of HIV.
The theory that a polio vaccine was the route for SIV to enter the human population was intriguing yet the only evidence to support it was circumstantial. In December 1993, Rolling Stone had to print a "clarification'' of its earlier story at the behest of the Wistar Institute in Philadelphia, which had made the Congo vaccine, and the scientist responsible for developing it, Hilary Koprowski. The central reason for the apologetic statement was that the committee of scientists had taken the case of the Manchester seaman into account in its review of Rolling Stone's theory. The scientists -appointed by the Wistar Institute - said in their report: "[The Manchester man] had returned to England by the first half of 1957, before the Congo trial was begun. Therefore, it can be stated with almost complete certainty, that the large polio vaccine trial begun in 1957 in Congo was not the origin of Aids.'' A Wistar Institute press statement in October 1992 re- iterated the importance of the 1959 case: "The most conclusive evidence refuting the origin of Aids theory involves the earliest documented case of HIV-1 infection - a merchant marine [sic] who was symptomatic in 1958 and died of Aids in 1959 in Manchester, England. "While this man travelled abroad to northern Africa beginning in Continues on page 3
From page 2
1955, he had returned to England by the first half of 1957, before the Congo trial was begun.''
However, it was the tenacity of one member of this committee - David Ho, director of the Aaron Diamond Aids Research Centre in New York City and professor of medicine and microbiology at New York University School of Medicine - that has now cast grave doubts over the scientific validity of the case of the Manchester sailor.
Professor Ho contacted the Manchester researchers in 1992 to learn more about the man, who had subsequently been named in the Sunday Express.
Professor Ho asked for samples to perform PCR tests himself. Manchester University's Gerald Corbitt said that after the Lancet letter of 1990 he and Andrew Bailey had tried to sequence the genetic code of HIV but had only limited success.
They had managed to get a partial DNA sequence - enough to know it was HIV-1 and not the other major type of Aids virus, HIV-2 - but had recognised their limitations. "To be perfectly truthful, we are a hospital diagnostic laboratory and we were beginning to get out of our depth,'' Dr Corbitt said.
Professor Ho's lab, however, was a specialist Aids centre and was accustomed to performing difficult PCR tests and rapid genetic sequencing. Soon after being sent processed DNA from kidney tissue - which had been left over from the 1990 experiment - Professsor Ho was able to isolate the entire sequence of HIV ``with ease''.
He did this in 1993 and now had the complete virus, from one end of its genetic code to the other. He also found that this genetic sequence was identical to the partial sequence of Corbitt and Bailey - scientific confirmation that it was the same virus isolated earlier by the two Manchester virologists.
The sequence, however, began to puzzle Professor Ho following a discussion he had with Gerald Myers, director of the HIV Sequence Database at the US's Los Alamos National Laboratory, in New Mexico, and a world authority on the genetics of the virus. "Gerry told us his concerns about the possibility that it was a contaminant. All the calculations and analyses Gerry did suggested that it could be a contaminant . . . [The virus] did not make any sense based on everything he has known about them,'' Professor Ho said.
Dr Myers was well aware from nearly a decade's work on the Aids virus that it is one of the fastest evolving life-forms. Its speed of change is dramatic. He estimated the strains of HIV circulating in the world alter their DNA sequence by about 1 per cent per year. This would mean the "1959 virus'' - which presumably must have infected Carr years earlier - should have differed from 1990 strains by 30 per cent or more.
The essential problem Dr Myers had identified is that the virus supposedly dating back to 1959 was to all intents and purposes identical to strains of HIV circulating in North America and Europe in 1990. "You couldn't distinguish it from a 1990 virus,'' Professor Ho said. Dr Myers dismissed the 1959 virus as an "aberration''.
Further evidence suggested that if this was a 1990 contaminant, it was no ordinary contaminant. For a start, Professor Ho had identified "quasi- species'' of HIV in the initial samples sent from Manchester. This means the virus he had detected was present as swarms of slightly different forms, indicating it was a genuine HIV infection with multiple copies of actively replicating virus. It could not be a one-off contamination.
Secondly, any accidental PCR contamination would be unlikely to result in an entire virus ending up in experimental material. Professor Ho was able to sequence the complete virus, which could only mean one of two things: either a complete clone of HIV had somehow got into the tissue sample or the tissue was genuinely infected with the virus.
The former is most unlikely, he said, because few laboratories use HIV clones (and Dr Corbitt's lab is not one of them) and in any case all sequences of such clones are known, and the sequence he determined was not from any known HIV clone in the world.
This left the New York scientists with an uncomfortable conclusion. "Given what we've done now in the past few months we would think the initial sequence was incorrect or there's been a sample mix-up ... We even discussed wild ideas that someone intentionally provided us with a sample that just came from a contemporary Aids patient,'' Professor Ho said.
In summary, he concluded that the initial sample of genetic material from kidney tissue sent from Manchester was genuinely infected with HIV but that this virus was disturbingly similar to 1990 strains. He faxed a note to Dr Corbitt in January 1994 saying how he was "greatly troubled'' by the sequence. Professor Ho was so concerned that he decided to ask the Manchester researchers for the actual tissue samples themselves, rather than processed DNA supposedly derived from them, to see for himself whether they contained HIV. After several months delay, in February 1994, he received a set of nine tissue batches from Dr Williams and Dr Corbitt. Each was embedded in their original paraffin blocks.
After an exhaustive series of tests using the most sensitive PCR tests available, however, he failed to find any evidence of HIV infection in any of the tissues, including kidney, throat, liver, heart, bone marrow, brain and pancreas.
As a final check, Professor Ho employed a sophisticated DNA test to see whether this set of tissues all came from the same person - they did. However, when he compared them against the DNA sent to him earlier, he was shocked to discover that this HIV-positive tissue was from another person. Furthermore, the size of fragments of a gene the scientists used as another check on their PCR technology indicated the two sets of samples from Manchester were from tissues of significantly different ages.
The HIV-positive tissue generated large gene fragments, a clear indication it was recent tissue, whereas the second batch of HIV-negative tissue produced small fragments, showing the DNA had degraded, as it does in older tissue. Everything pointed to the positive batch coming from a 1990 Aids patient.
The 1990 Lancet research had therefore failed the ultimate scientific test of its validity: replication by other scientists. It will now have to be retracted. The tissues of David Carr appear after all to have been HIV negative and his fatal illness the result of another, unexplained cause. Mr Carr's condition remains as much a mystery today as it was in 1959.
Dr George Williams: the pathologist
Dr Williams performed the post-mortem examination on David Carr that determined the cause of death as pneumonia. The first-named author of the 1960 Lancet paper describing Carr's mysterious medical condition, he later discovered Carr's stored tissues samples and presented them for analysis by Manchester University virologists. He retired three years ago.
Dr David Ho: the Aids scientist
Head of the prestigious Aaron Diamond Aids Research Centre at New York University Medical School, Dr Ho is considered one of the foremost Aids scientists in the world and helped to unravel how the Aids virus causes death several years after infection. Dr Ho performed the detailed analysis of the tissues samples from David Carr and found them to be HIV-negative.
Dr Gerald Corbitt: Manchester virologist
Dr Corbitt is head of the virology unit at the University of Manchester Medical School. Along with his research assistant, Andrew Bailey, Dr Corbitt performed the analysis on the tissue samples sent by Dr Williams which found they were infected with HIV. The three researchers wrote up the results in the Lancet in 1990 and received world-wide acclaim.
Dr Trevor Stretton: the hospital doctor
Dr Stretton and his colleague Dr John Leonard were the two doctors in charge of David Carr on his admission to hospital in 1959. They were co- authors, with Dr Williams, of the 1960 Lancet paper that described Carr's illness. Neither was involved in the subsequent tissue analysis that led to the 1990 Lancet paper. Both now retired, they keep an active interest in the case.
Dr Gerald Myers: the HIV analyst
Head of the HIV Sequence Database at the Los Alamos National Laboratory in New Mexico, Dr Myers is considered to be the foremost expert in the study of HIV evolution. It was Dr Myers who first alerted Dr Ho to the unusual nature of the virus that was isolated by him and the Manchester researchers supposedly from the stored tissues of David Carr.
Andrew Bailey: the lab assistant
Mr Bailey carried out the key benchwork on the tissue samples sent by Dr Williams from the pathology department. He works under Dr Corbitt and repeated his analysis twice in a ``blind, controlled'' experiment involving 12 samples, six supposedly from David Carr and six from a HIV- negative control - a man who died in 1959 in a traffic accident.
HIV: the virus
The virus first came to prominence in the early Eighties and is now on track to become one of the biggest killers of young adults, especially in Africa and South East Asia. It has always been a mystery as to why the virus should have apparently suddenly appeared in the latter half of this century. The dismissal of the 1959 case now leads scientists to the conclusion that it is indeed an extremely recent virus to emerge in the human population from an ancestral virus that infected monkeys, man's closest relative in the animal kingdom.
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