Resistance and Resistance Tests

Professor Clive Loveday

CAB 2: Friday 2 Aug 2002

This is a transcription of the training session on genetics, resistance, resistance tests and HIV that was given to the UK-CAB by Professor Clive Loveday. The full set of 60 slides to accompany this talk are available on the i-Base website with the other reading material for the August 2002 meeting.

The talk provides an overview of these issues in non-technical language against the background of HIV research over the last 15 years. Transcription edited by Simon Collins.

Introduction

I started working in the epidemic in 1984 and I worked as a clinician looking after patients in London for about four years at the Middlesex Hospital. I then moved into laboratory research and became a medical virologist (someone who studies viruses) and looked after the HIV section as a consultant in that hospital. In 1996 I became the first Professor of HIV/AIDS in the UK, running a department in the Royal Free for seven years. Essentially there was a political change within the medical school and as such they were not really keen to support a department of HIV/AIDS but rather wanted me to amalgamate into general virology. The profile of previous work there wouldn’t have continued and so I decided to continue this with a new charity. I now continue to support and be supported by all the groups I was working with essentially except the medical school.

The International Laboratory Virology Centre (IVAC) is a registered trust and is a not for profit organisation which is very unusual and may be the only organisation of its kind. We have offices, laboratories and a teaching centre just west of London and a staff of ten.

The objectives of the charity are around developing and applying real time, quality assured, economic molecular biology assays to enhance patient care. We collaborate with 41 district general hospitals around the UK and also continue to do the lab work for the Royal Free. We also support clinical trials and audits and again these are many of the groups we used to work with. We contribute to international studies improving understanding of patient management including the MRC studies FORTE (comparing 3 and 4 drug combinations), ERA (Evaluation of Resistance Assays), PERA (Paediatric ERA). EuroSIDA and SPREAD are two European funded studies and INITIO is a large international five-year study looking at benefits of starting with or without protease inhibitors. We are working with the virology and I am on a number of the committees associated with this work. We also have collaborative grants from industry, most of those are to do with improving and assuring molecular technologies for patient care.

One important thing to remember this morning is that this virus is always evolving and there is always the possibility of it evolving away from any current test so that we cannot detect any more.

In addition to that we are looking at a programme in developing low-tech molecular approaches in the developing world. 90 per cent of the people in the epidemic do not benefit from therapy at the moment and as well as treatment they need to be supported if possible by the sort of technologies we take for granted such as viral load, CD4 and resistance tests..

Finally, our last object is teaching to help all groups from consultants to community groups like yourselves understanding new technologies. Also in the teaching section our charity has taken on the role of training laboratory technicians in understanding HIV technology.

Introduction to genetics: genes and DNA

Before discussing HIV drug resistance testing I have written a pre-section about understanding genetics. This will make sure you understand the language associated with one of the biggest subjects today. Genetics is always in the news on TV and in the press – everything is moving towards genetics and genetic manipulation and understanding those processes. Trying to understand genetics is an important before we even look at HIV – it is the bread and butter – it is the way of communicating in terms of what we do with HIV.

The human genome (or genetic code) carries all the information about an individual in every cell in our body, with the exception of red blood cells, and it is in the form of a chemical package called DNA.

The information is packaged in what are called genes and these genes are collected in a group, or a mass, in what are called chromosomes; different genes are different sizes and code for particular features. Lots of them are packaged in larger chromosomes and there are 23 chromosomes in humans. This number 23 is arbitrary. If you look at another species you might find that they only have 21 chromosomes so don’t worry about that number. Chromosomes as an element are somewhat disappearing in discussions and are being super ceded by DNA sequence.

There are about 30,000 – 80,000 genes (or packages of DNA) in humans carrying all the genetic information required to build another person. Interestingly enough those genes, that genetic information only represents about 10 % of the total DNA in cells. In other words 90 % of DNA contains either non coding archived information or other junk that we just don’t understand at the moment. For instance, old retrovirus sequences have been found hidden away in this dud DNA which showed that at some time in the long distant past, the human species has been infected with an HIV-type virus before. This is why there are fragments of a previous retrovirus DNA within humans.

There was a lot of media coverage when on June 26th 2000, scientists ‘published the human genome’. This meant that they understood the rough chemical sequence of the human DNA. They had taken out the DNA, and starting at the beginning they continued until they sequenced the whole chain. This provided the chemical sequences of the whole of the DNA – but not what all the individual genes code for. So although the genetic code has been broken, that is very different from understanding what it does.

So far only 10% of genes or packages of genetic information have been identified as being associated with certain elements in the body.

Every cell contains two complete sets of genetic information for the human genome. This is the so-called double stranded DNA. So there are two strips of DNA that are wound together in a helix. The exceptions are eggs and sperm cells that only carry one set each for obvious reasons. Each cell therefore contains 30,000 – 80,000 genes, with all those discreet units of genetic information on 23 chromosomes. When we breed we pass on one set of each and then there is a process of swapping this maternal and paternal genetic information called recombination. That takes place and ends up with a new individual when it winds up as a double strand of DNA again and that new individual will carry elements of the parents from which the egg and the sperm came. So that is kind of how the chemistry works.

DNA – a book with letters, words and paragraphs…

You can imagine the human genome as a book:

• For humans this book has 23 chapters called chromosomes
• Each chapter has several thousand sentences, all telling different aspects of the story, called genes.
• Each story is made of paragraphs called exons and these are bits of DNA that encode for something important
• Each paragraph is interrupted by advertisements called introns and that is great spreads of dud DNA that as far as we understand, appears to do nothing.
• Each paragraph in the stories is made up of words called codons.
• Each word is written in letters of the alphabet, called bases (and there are only 4 letters in the genetic alphabet)

There are one billion words in this book and if you read one word per second (ie reading the genome one codon at a time) for 8 hours per day it would take a 100 years to read the whole of the DNA sequence. So there is this gigantic document in every microscopically small cell.

The genome, like a book, is written in a linear one directional, one dimensional form and is defined by a code that transliterates a small number of alphabetic signs into a large lexicon of meanings because of the way that they are grouped and ordered.

Just the same way a book will transmit and give us a broad feeling of what is going on within a story, so the genetic code does exactly the same thing.

Bases, amino acids and proteins

In chemistry everything is made of four chemical ‘bases’ which are given the letters A, G, C and T. They are adenine, guanine, cytosine and thymine and in different sequences they form a code. And the code is all about proteins and almost everything in the body is either made of protein, or it needs protein to make it.

Proteins may be structural, and help to build the body, muscle, parts of the bone; they may be non-structural; or they make enzymes which are particular chemicals that facilitate reactions to build sugars or fats that we also need for the body to work.

Proteins are made of chemicals and the building blocks for proteins are called amino acids. These are quite small chemicals and there are 21 ‘essential amino acids’ that we get in our diet. We eat them and we absorb protein and these are then broken down into amino acids that are used to build new proteins.

Each group of three base spells out a code for a different amino acid and it is the order of the bases determines what the amino acid will be.

So now you can see DNA is a template for building proteins.

How does DNA copy itself?

For life to continue from one generation to the next – whether for humans, animals, plants or a virus – relies on an accurate way of copying these proteins.

This means that DNA has to replicate (or the book has to photocopy itself). This is possible because of a unique property of each of the four bases to only bind to one other type of base:

• A will always bind with T
• G will always bind with C

Figure 1 (slide 10) below shows how this works.

There is some replication going on. Although the bases of two genetic messages within one cell look random, remember that they are broken down into groups of three bases and each group of three bases is a code for an amino acid.

When this replicates an enzyme in the nucleus splits the double strand open and just adds the opposite base, as you can see, to each side. So what you are knitting down the middle of the DNA is a new faithful copy. You now have two faithful copies of the genetic message in the cell that is split in half.

Making proteins

The making of proteins is a little more complex and I’ll take you through that as it is important. It involves translating the genetic code.

First the code is transcribed by copying the base pairing as a chemical called RNA, which is very similar to DNA, but is a single strand. In RNA there is a base called uracil (U) instead of thymine (T).

DNA is trapped inside the nucleus. In comes RNA – and this is called messenger RNA – that also carries a linear code but it is much shorter. Messenger RNA gets into the nucleus, it can create a faithful copy of a part of the genetic code, let’s say one gene, a much shorter length, and then it can leave the nucleus which DNA can’t.

So you see what you have got is a bit like a courier. It goes in and makes a copy of the bit it is interested in and then carries the code to the cytoplasm. It carries the code back to the cytoplasm because the protein-making factories (called ribosomes) are there. The messenger RNA translates 3 bases at a time, that is one codon, and the amino acid is transferred by another sort of RNA that is only found in the cytoplasm, called transfer RNA, according to the codon present on the messenger RNA. It all sounds very complicated so what I have done is to give you two representations of this …

The genetic message in the nucleus – each one of these triplets, so called colons, which is 3 base chemical codes for a certain amino acid but it is stuck in the nucleus – the messenger RNA makes a faithful copy of this in the nucleus and here is the faithful copy – here are the Us instead of the Ts – I told you they will always bind G to C or A to T, or to U in this case. This messenger RNA can then move to the cytoplasm and it fixes itself in the ribosome and the ribosome is a knitting machine which knits protein basically, and as this messenger RNA moves across the surface of the ribosome then transfer RNA is a special modified RNA which carries the appropriate code at the top – and then has a small body and it carries the amino acid at the other end.

The process for this is:

• the nucleus has the DNA inside it with the genetic code
• one bit of the DNA is being translated,
• the messenger RNA is faithfully copied from this piece of the code
• it can then move into the cytoplasm.
• The same messenger RNA then goes through a ribosome (which looks like a figure 8)
• as it goes through the middle where the enzymes are you get these small chemicals called transfer RNA and each one carries a code at one end (three bases) and it has the appropriate amino acid stuck on the other end
• as this moves through the machine the amino acids are bound together (so you get the RNA moving through the ribosome and then parallel to it you start to knit one amino acid to another and another and you end up with the protein that will contribute to structure or be an enzyme or whatever.

So that very simply is how genetics works!

You should now understand the language, the difference between DNA and RNA and simply what it does inside a cell and why it is trying to do it. There is no mystery – a cell is trying to make protein and that protein will help to build or help to facilitate the building of a new body.

Q: What does RNA and DNA stand for?

A: RNA stands for Ribonucleic Acid. DNA stands for Deoxyribonucleic Acid. There are plenty of books about this but you now understanding the principle of what this system is trying to do. Also the process associated with the synthesis of new proteins is quite well understood now and that code and process was understood very soon after Crick and Watson discovered what DNA did and how it was made in 1953.

Q: How do we find out which bit of the DNA code for what?

A: There are lots of different ways of doing it but for instance if you have someone who has a deficiency of one protein you would look at a piece of the genome versus other pieces of the genome. That would give you a focus where you could find a difference. If the person without the protein has something missing in their genetic code you could assume this must be the area where the gene is that makes that protein. But you still don’t know where the gene begins and where it ends. Then you need to explore that area. If you assume it is 500 amino acids long you can use computers to move that up and down that area of the genome and see where it fits.

Q: You said that 90 % of DNA is archival … is that just a guess?

A: No, that is understood in broad terms. It is a guess down to the last base or two but it is that sort of proportion because we know what 8,000 genes do and we know roughly their average size and we know how many genes would be needed roughly to build a human body so we calculate but there is far in excess of the DNA required to code for that. Also by exploring different parts of the genome we can find junk, bits of retroviruses, also premalignant factors that code or switch on issues around cancer – so cancer research is exploring this.

Genetics of HIV

The next series of slides will just be an introduction to HIV so that again we are all using the same language and we all understand issues around what HIV is and what it does.

• June1981 – the first five reported cases of Aids came from San Francisco and then New York
• 1982-83 understood to be occurring predominantly in homosexual men, people receiving blood transfusions and blood products, and also intravenous drug users and prostitutes. This was a similar picture to hepatitis B the decade before and therefore we were looking for a sexually transmitted/blood borne organism, probably a virus and that led the search to look for a virus.
• 1984 – first serology (HIV) tests were used in the UK
• 1985 – the full gene sequence of HIV was mapped, again it is the same as the human genome, someone had read it from beginning to end but knowing what it all did is a different question.
• 1986 – use of first antiretroviral drug, AZT. At the Middlesex I ran part of the first AZT trial in the UK (the two other centres at St Marys and St Stephens).
• 1987 – heterosexual spread was identified (we were part of describing that in the UK).
• 1987-88 – PCR technology was developed – PCR stands for Polymerase Chain Reaction and it is a way of chemically manipulating genetic material so that we can make tests to look at genetic information rather than the products associated with it. I’ll go through what advances have been made with this and the shortcomings.
• 1989 – antiretroviral resistance described by Brendan Larder and Doug Richmond.
• 1990 – first viral load assay for clinical care (using PCR technology).
• 1991 – first resistance assay made at Middlesex hospital using PCR technology.

In 1991 we went to the MRC and asked for a grant to validate these tests and we sat before a committee that concluded that there is no way they could ever see a virological assay measuring viral load being of use in the clinical care of patients with HIV. I wrote that down in my diary because we were shocked. One of the major reasons for that was that they said to us that it would be individualising patient care and that never before in medicine have we had individualised patient care! We said, ‘Rubbish, you are going to have to individualise patient care because they all have individual viruses’ – but we couldn’t get the message across.

In fact we didn’t lose because there were many drug companies that came to us. They’d seen what we had published on the viral load assay and they discreetly slipped us little boxes of samples and said ‘Could you please test these and see what is happening?’ Often they didn’t tell us what they were and it was obviously their monotherapy samples. We tested it and gave the results back for obscene amounts of money that we then used to validate the assay in the long run – so we didn’t need the money from the MRC.

It was curious because we always knew. Some came back and said, ‘That’s interesting – we’ve got another set of samples that we want you to look at’ and others would say, ‘We don’t understand this, we don’t think this assay of yours works’. So you could spot the drugs that were not going to be hitting the market!

• 1993-94 commercial drug assays became available and we moved over to these because making your own assay and quality assuring all the agents on a regular basis is an enormous laboratory task
• 1994 – the dynamics of HIV replication was defined by our group and by other groups.
  We plotted changes in viral load and that in less than two days you had dropped viral response by 50 %. This gives you an idea of the dynamics of HIV replication and we had never understood this before. Up to that point in time we were saying to patients – you get infected, have acute infection which causes your immune system to react, but then you get a dulling down of replication and for years and years the virus is not turning over at all or was very slow. None of that was true. We actually realised that throughout the infection virus is replicating incredibly rapidly throughout that time but the body is handling it in a different way, the body is succeeding in clearing it.
• 1995 – viral load became recognised as a study end point (from the Mellor’s data).
• 1996 – PCR-based commercial resistance assays were produced.

Figure 2 (slide16) is quite a good electron micrograph showing the structure of HIV.

It is about 100 -110 microns across and so you get about 70 of them across a pinhead. The core carries the genetic message. HIV carries its genetic message as RNA and it is kept in the little protein core. Then you have the envelope (outer coating) of the virus. HIV is a double lipid (fat) envelope which you can see quite nicely in some of these viruses and plugged into that double lipid envelope are the proteins of the envelope, proteins of the virus. They look like little mushrooms and I am sure you’ve all seen representations of this.

There are different shapes because you see cut sections at different angles. The cone section is the side – if you cut it another way it looks circular. The cone or coffin shaped capsule is usually shown in diagrams.

Figure 3 (slide 17) is from an early viral load assay that we did from patients receiving AZT in 1986. You can see the response (-3 to – 4 log reductions) from monitoring AZT monotherapy and the waning of that response over 24 weeks. That is an archival slide and I think we published that in about 1990 using our own viral load assay.

Q: You saw this in 1990? – where it is clear that you get a response and after 6 months it has gone. Why were so many people kept on AZT monotherapy for years? Didn’t people understand viral dynamics?

A: This sort of curve from about 25 patients’ was used to define these early dynamics. It is very difficult to actually get people to hear you sometimes. You can publish it, you can shout quite loud, or you could get your face on the front of Time and everyone hears about it immediately. We never quite networked in the same way. We were doing this in 1990-91. By 1991 we had done a resistance test here and found there was resistance as Brendan Larder had described and we published that as well. From the time this work was done it took two years to get this published – we presented it at meetings but it wasn’t until early 1995 it was published in the Lancet.

We were curious about all the movements in the above curve – and we did this one particular patient three times and we got this shape every time – so these sort of movements are probably real in terms of the assay.

You have to remember the pressures everyone was under. First of all as far as patients were concerned there were two choices- AZT or nothing. People who were on it were waiting for the results of the Concorde and the American 09 trials and so the choice was stay on it or don’t stay on it. You heard some really curious things said – AZT is azidothymidine and I heard physicians say, ‘Well it is not going to do any harm, it’s like thymidine and you’re eating thymidine every day’. Excuse me but there is a great big N group on the end that we never find in our diet – it is found in nerve gasses but nowhere else!

You fight very hard – as we did with the viral load assay – the MRC panel was establishment and we were going to them and you are trying to present a completely new way of looking at something and basically we were blanked out.

Q: Did you say you had that in mind in 1986?

A: No, in about 1990. 1986 is when I did the AZT trial and I was looking at immune complexes at that stage but we had stored and kept all the samples from that trial at minus 70 degrees. You have to have blood samples that have been quite well stored if you want to measure those sort of measures with viral load so coincidentally I happened to have the first therapeutic cohort stored in the right way to measure viral load on, so we were one jump ahead.

Q: AZT wasn’t a new drug?

A: AZT was about 20 years old and it had been trialed by Welcome as a potential anticancer drug and had been shelved as being far too toxic ever to give to humans.

Q: Why was dosing so high?

A: Yes, in that first trial we were giving 1500 or 2000 mg per day. That trial was not focussed on clinical care. You discover these things afterwards but basically an MP asked a question in the House of Commons, about why people in America were getting treatment for HIV but not in the UK. Within three months we had been recruited with three other centres – I can tell you this now because it was so long ago. We received the drug from America, not in tablet form but as intravenous infusion form. The whole of that trial was run with the drip form of AZT made up in orange juice. The dosing was a complete guess – much higher than is used now. What is interesting is that we got a 3 or 4 log reduction and it shows some sort of dose response that is possible with AZT because we end up using 500 or 600 mg per day now.

Q: Higher dosing was used in the capsule form as well.

A: Yes, the Concorde trial was using 1000mg I think and the American equivalent which was about six months ahead of us was using an even larger dose. Deciding doses is not a precise science and it is driven by the pharmaceutical companies, we just do our best. The other sad thing about that trial is that we had been looking after our own cohort of patients. This was a natural history study at that stage – people who had HIV infection and we were supporting their clinical care.

We had always said that whenever a new treatment was discovered the longest servers would be the first to benefit from any therapy. This was philosophically the right thing to do but what we ended up giving our sickest patients the highest doses of AZT. A lot of our patients had less than 50 CD4 counts and they showed all the side effects as they were more vulnerable. The same doses in people who are very well wouldn’t show the same side effects.

We were only given six patients each and it actually took me eighteen patients starting the study to get six through three months treatment because of the toxiciy. About 18 patients were treated for 3 months and then the minister of health stood up in the House of Commons and said, ‘I can now tell everyone in the House that the new cure/treatment for HIV is now available in the UK and is being used.’ It was a political game.

Q: Is there a difference in understanding application of genetics between HIV-1 and HIV-2?

A: No the principles would be the same, but there are structural differences, for example they are very different in the envelope.

By 1985/86 or a bit longer we had defined the genome for HIV – and this is shown graphically in Figure 4 (slide 18).

Instead of billions of bases (as the human genome has) there are 9,200 bases for HIV. So it is a very small genetic code in relation to the human genome. Over time we evolved where the genes were that coded for these proteins and each of these proteins (called ‘p’) just means protein. p-24 means protein with a molecular weight of 24,000. If you see gp- used, which you find in the envelope it is because it is a glycoprotein – it has lots of sugars on it. So gp-160 is a precursor protein of 160,000 molecular weight which is then cleaved by protease to give two proteins the gp-120 and gp-41 which make up the cap and the stem of the mushroom which forms the envelope of the virus.

A lot of these little proteins – the VPR – produce very small proteins that help control replication. They were all to do with the success of HIV, this virus was able to control whether it was replicating or not within cells.

The three structural genes overlap. There’s something else very clever here which just amazes me. There is one length of 9,200 bases that are all in a line, but it has three open reading frames.

What that means is if you start reading the genetic code in one place you’ll make a certain protein, and if you start in a different position reading the same bases you make a completely different protein that has a completely different function. You can read along there three times and pull out different proteins and I don’t understand how something that complex and clever ever evolved…

The replication of HIV involves a process called reverse transcription and the virus’ genetic message is RNA (one strand) – so for this virus to infect a cell it has to effectively make a DNA (double-stranded) copy of its virus. This is a complete reversal of what humans do to make proteins and that is why it is called ‘reverse’ transcription.

Retroviruses are the only group of viruses that do this – hepatitis B does to a degree but it is really only retroviruses – and hence their name. They convert their genetic message in RNA to DNA to then integrate it in the nucleus and keep a message of the virus in the nucleus for as long as the host is alive.

The important thing about the reverse transcription process, which is essential when a virus is infecting a cell, is that it is prone to making mistakes. These are called ‘base errors’ where it puts in at least one wrong base in by mistake. Now because HIV has such a rapid turnover – one generation in one and a half days – the process of base errors is a route for HIV evolution.

Those mutational changes are taking place every day and they may influence the virus outcome in a number of ways. Mistakes happen much less frequently in humans because we have a second enzyme which goes along behind correcting mistakes. HIV though is a far more simple organism, and does not have a corrective capacity.

In HIV these mutations may be:

• neutral, ie they have no influence on the biology of the virus whatsoever, so they will just be there, just appear and be there.
• may make the virus less fit, ie a mutation may make CD4 binding less successful. This is a distinct disadvantage to the virus and then you would get poor virus survival in a virus that has that difference.
• it may be an advantage, the mutation may confer some benefit and improve the virus survival.

In humans. even small base changes can have a huge impact. Cystic fibrosis is a disease which leads to enormous changes of protein secretions which damage the lungs and the pancreas, and the survival rate of people with cystic fibrosis is very limited. Cystic fibrosis is due to only three or four base changes in the DNA.

With HIV replication, out of a hundred new viruses that bud from a cell probably only one is complete and infectious, the rest are rubbish. That is important to remember. But even 1 in 100 replicating going out to infect another cell is enough.

Viral diversity: quasi-species and sub-types

In an HIV-positive person there are almost-daily rounds of replication. So with a mistake in almost every replication you are getting a continual expansion of the types of different viruses. This is called genetic diversity and the longer someone has been infected the greater diversity of the virus. So you may be infected with one virus but within a year you actually have a whole population of slightly different viruses circulating, which have all evolved from that one virus. These slightly different viruses are called the ‘quasi species’. The majority virus is bigger, stronger, and more successful at replicating, and genetically slightly different ones are less common. Virus’ with the most changes tend to be much less frequent but even one virus may carry the resistance to a drug.

If that drug is then taken by the individual this rare virus with be relatively stronger and rapidly becomes the new majority virus. Generally, one to two years after infection an equilibrium is achieved where the stronger virus continues to replicate.

Q: Once you understand you have lots of different viruses inside you, how do the tests work and how do you know they have taken a representative sample?

A: Because we look at the majority virus and also because of the genetic diversity in a host – intrahost diversity – is such that they are all very closely related so if you looked at all of the viruses in one individual, less than 1% difference in their genomic structure.

Geographically a broader genetic diversity of viruses exists around the world. These are called sub-types or clades. In this case virus populations can be so genetically distinct that we characterise them there are now around ten different subtypes that be 30% to 40% different in their genetic structure.

Certainly the commonest subtype is B and contributed initially to the epidemic in the developing world. Subtypes A through to O are found in different geographical areas. A lot of these focussed initially on sub-Saharan Africa but with air transport, and population movement, in exactly the same way as subtype B moved, these subtypes are now moving as well.

Compartments and sanctuary sites

We tend to assume that it is the same virus and the same amount everywhere in any individual. That is not true and the distribution of HIV in the body is not all the same.

We usually just think of the blood compartment – because this is what we can test easily for viral load and resistance – but only 2% of you HIV is the blood. The other 98% of virus is in different compartments, from the lymph nodes, from the liver, from the spleen, from the neural tissue, and they are all producing virus, possibly at different rates.

HIV overflows from these compartments into the general blood circulation.

The other half of the dynamic is that it is continually being removed by the body, normally by some sort of immune modulation which takes place in the liver, the spleen, or other sites.

So you have a continual production shown in figure 5 (slide 25) and a continual removal – and amongst that dynamic we only usually measure what is in the middle.

A good example of the importance of this is somebody who has their spleen removed. Because the spleen removes HIV, the day after a splenectomy, their viral load would go up by a log – so it is an important dynamic that is very sensitive to change in any of these elements. If a patient suddenly develops liver failure, an important way of clearing his virus stops working in this way and viral load will go up.

Equally rates of production may be different and certainly from our work, and Doug Richmond’s work, we have looked at resistance to viruses in different compartments, it can be completely different – turnover rates are different in certain tissues and therefore evolution is different also so you may get resistant viruses which are many generations behind those which are in other compartments.

Generally, we measure resistance in the blood, which is a mixture of everything, but we may need to know about resistance in other sites. We use consensus sequencing, so we are always looking at resistance in the majority of viruses, but the majority virus isn’t always what we are interested in. Minority viruses may be the ones that cause trouble later on if the environment changes.

PCR technology – How a viral load test works

PCR has fundamentally and completely changed the laboratory management of patients with HIV. It is called Polymerase Chain Reaction and was developed by Kerry Mullis in 1985. It is basically a process of using a machine to reproduce specific DNA sequences.

We are now able to make DNA in the test tube. I showed you a replication of DNA in a cell and we can do that now in a test tube. The technology is possible because of computerised advances with new machines that allows tiny detection of otherwise undetectable specific microbial DNA.

It has to be specifically primed, amplified and biochemically detected and because we have this technology we now have diagnostic tests: you can take a bit of saliva, mucus or blood, you can extract the DNA from it, you can put it through this process and in four hours you can know if there is DNA there from a whole number of micro-organisms.

Clearly before we were trying to grow viruses, trying to grow bacteria, looking at serology, looking at antibody changes to them… most of that has been sidelined now because this is like real time detection of very small quantities of microorganisms. It requires heat sensitive enzymes, an automated primer sequencer so we are actually making nucleic acid to carry out a reaction.

It needs a thermal […] which is a computerised driven box which will take you through a whole series of reactions and each reaction just takes a couple of minutes.

You start off reaction one – you’ve got two double stranded nucleic acid, DNA, and the machine splits it and replicates it in two minutes – you’ve then got four copies – and it will go through a cycle every two or three minutes so by the time you get through thirty cycles of the machine you’ve then made thousands of millions of copies of a piece of targeted nucleic acid.

Now these two copies are totally undetectable in biochemistry but if you get a thousand million copies we can detect that using standard biochemical techniques. We look at bands on gels or we can produce a reaction to it, and get a colour reaction. So really the PCR reaction has revolutionised the way we diagnose and manage patients with HIV. It is also vulnerable and I will talk about that vulnerability later on.

How did we apply this technology?

This technology led to diagnostic assays, quantitative viral load assays and resistance assays, both phenotyping (looking at the biology of viruses) and genotyping (looking at the sequencing of viruses). Testing for antibodies to diagnose infection still is a cornerstone in adults for diagnosing infection but most of the other tests that involve management of patients are now PCR based molecular assays.

Drug resistance testing and supportive therapy has been used in infectious diseases for years. It has been widely used in bacteriology and we use that to guide antibiotic selection. In virology it was limited by lack of technology but basically drug resistance at the high frequency of replication I talked to you about allows evolution of HIV specifically in reverse transcriptase and protease where our drugs act.

This is what resistance is. It means the enzymes that HIV drugs should disable, can continue to work in the presence of drugs. The different viruses we are talking about are also archived in an HIV-positive person and may remain there for many years and evolution of resistance represents an important cause of failure.

There are basic rules that hold true for HIV and resistance:

1. with rapid replication you have HIV evolution day by day
2. that evolution obeys Darwinian rules, that is things in the environment that will drive the evolution, and fitter viruses will form the majority population
3. HIV responds rapidly to environmental pressures – ie if you add a drug that works against some virus but not other virus, the virus that is unaffected (or resistant) will become the majority
4. iv) Single mutations occur every day in untreated patients. Double mutations occur less frequently but they do occur.
5. These mutations give no advantage to the virus, in fact they probably give a slight replicative disadvantage and they remain the minority population.

You also have to remember as well as evolution of drug resistant viruses there are other causes of drug failure, always a message to pass on, first of all the pharmacological issues around absorption, distribution, metabolism of drugs, interaction, activation, and just as importantly – adherence to therapy. If you don’t have adherence to therapy then you are clearly going to be giving suboptimal doses and you will evolve resistant viruses.

Resistance tests – what is measured

Resistance is measured in different ways by two different approaches.

Phenotyping is where we look at the capacity of HIV to grow in the presence of individual drugs. We take virus from an individual, we grow them in increasing concentrations of drug and we look at the way they are able to grow and therefore see if they have resistance. This is a biological test.

Genotyping is a completely different approach where we sequence all those bases that make up the genetic message in the reverse transcriptase and in the protease part of the HIV gene and we look for changes which we know to be associated with resistance. This is a molecular approach.

Phenotyping has been done by microbiologists with bacteria for thirty years: growing the bugs in the presence of the drug and seeing if they are inhibited or not by the drug – we now do the same thing with viruses. There are really three ways for doing that. One way is to culture (grow) cells from a patient with naive cells from the blood transfusion unit and look at their capacity to grow in the presence of drugs.

Virco and Virologic are two companies that have made a slightly different approach. They take the viruses from the plasma of the patient, then reverse transcribe it and then amplify it, so you then have an amplified population of the patient’s virus. This is then ‘shotgunned’ randomly into recombinant viruses – viruses that don’t have an RT in them already. Those recombinant viruses are then cultured in the laboratory in a standard system in the absence and presence of drugs. It takes about three weeks and you can see if the viruses from the patient are affected by different drugs.

With genotyping, a number of companies that have taken the initiative to modify sequencing which we have been doing for years and years in the laboratory. Sequencing can involve the whole section of a virus or to just look at very specific mutations – ie you could look at the whole RT section or just for mutations associated with resistance to AZT and 3TC. Our first resistance assay in 1990 was just such an assay.

Advantages and disadvantages for each test are shown in figure 6 (slide 38).

Genotyping is a technology that is available in many labs, it is rapid, it is not technically demanding, mutations associated with drug resistance may precede biological or phenotypic resistance and it quantifies a proportion of wild strains so you can use it to get some sort of semi quantitative measure of the proportions of wild and mutants in a population. Disadvantages include that it is an indirect measure and it has a limit of detection [sometimes down to 2% – 5% in research but for normal sequencing probably just about 20 %]. It may not correlate with phenotype for various reasons, complex data is generated and it needs interpretation. It is an expensive process relatively and there may be difficulties with interpretation.

Phenotype is a direct measure. It is a very familiar technology to physicians who have been doing bacteriology for years where it has proved to have a clear clinical relevance. However the limitations are that it has a restricted availability, it is a slow process, it may take weeks to do the experiments. It is technically very demanding – you need a category 3 laboratory and there are only two or three sites in the world that do it in factory type conditions, so we have to send our samples to them. It is very expensive and less sensitive than sequencing and it doesn’t look at sensitivity to combinations of drugs. You’ll always get reports that just give you sensitivity to individual drugs and whether there is any cross influence we don’t know at that point.

Slide 39 shows how Visible Genetics have modify the technology. This shows that we now use tiny little 6″ x 6″ plates. When we started doing this freehand in the lab twenty years ago we were using plates which were about 1’6″ x 1’0″ – there is an enormous advance in the technology and safety. Towers run the sequences and read them and they then immediately download onto a computer. So they have taken the principle of sequencing and turned it into a system which we can use in the clinical la

They also have what is called a gene librarian shown in figure 7 (slide 43).

Each one of those squiggly lines represents a base. Eeach colour is a different base and so you can read these and see that each of these peaks gives you a readout. Each base relates here to the codon (printed about the lines) and we know what they stand for and which ones we are interested in.

The genetic message is in two strands, two copies – and the computer it reads and compares it in both directions. Then you have the opportunity to read through in the gene librarian and make a manual review of the whole thing. Those are the sort of squiggly lines we look at all day long…!

You need rules to understand which mutations are important and that all this is done in an experimental way and new research is regularly published to undate this understanding.

What you end up with is a set of rules showing which mutations, or which changes in which codon will contribute to resistance to each drug. Slide 46 and 47 show the International AIDS Society tables for NNRTIs and PIs. But to actually be able to interpret these a number of computerised and panel interpretations come along.

So the gene librarian has a panel of experts who go to all the conferences and look at the evidence for different mutations and will add it to the gene librarian and decide which new study results are important and good evidence that certain mutations affect resistance.

Q: Does this include all possible mutations?

A: No. I am on the IAS panel and we marked major mutations more heavily (called those primary mutations) and others we called secondary mutations which are written in a finer line. We tend to drop the terms primary and secondary now and tend to call them major mutations and minor mutations.

Essentially what is happening in terms of the protein is a major mutation is a mutation that results in changes in the protein structure for the enzyme (usually at the active site of the enzyme) in such a way that the drug no longer works. Minor mutations are usually areas of the protein around that produce configurational or slight structural changes in the protein that help it to bind more easily to its target and facilitate the action of the enzyme.

That is in principle what we are doing with sequencing. There are different groups, different interpretation mechanisms and different panels but it is important that they are kept up to date all the time.

Slide 49 shows an old antivirogram report to show you one standard way of interpreting phenotypic resistance, ie biologic sensitivity. In this system Virco chose to list the drugs and define a sensitivity as 0.1, one, tenfold, hundredfold, thousandfold change in sensitivity. So if you then look at this individual report you see a 35 fold change in sensitivity to zidovudine. Colour coding s then used to show resistant, intermediate and a sensitive.

The main reason this changed is because the same cut off values were given for each drug. Up to four fold was always regarded as being sensitive, four fold to tenfold was thought to be intermediate and above a ten fold change in sensitivity was always thought to confirm resistance.

We now understand that different drugs have different cut offs, and certain drugs like dT4 may require a much lower change fold in sensitivity to confer resistance and to have some impact. Nevertheless, in more recent reports the principle is the similar, and colour coding is used to indicate sensitivity and resistance and some sort of dynamic bar chart is produced for us to read off and to show physicians.

Q: Have cut-offs now become more specific with some of the drugs.

A: Yes, we are now defining the cut offs more carefully.

Q: And I saw a table where some of the drugs it had a cut off of ten – are these now accurate?

A: This is not something that companies generally want to spend a lot of time doing. The thing that drove BMS for instance was the fact that ddI and d4T were giving anomalous results in combination therapies and there was a need to try and find what the sensitivity. Plus people like Virco were exploring that in their studies. It is an issue of what we call genetic robustness, certain drugs like AZT here go up to a thousand fold, one mutation will produce a thousand fold change in sensitivity.

A vulnerability of phenotyping is around determining the real cut off that has implications for patients. For genotype sequencing it is around expert panels keeping up to date with the data and revising rules panels every four or six months which is what happens.

Virtual Phenotype

Another area of activity that Virco carried out is called a virtual phenotype. This takes the sequence (genotype) data from a patient and compares it with a large computerised database of existing sequences with a known phenotype – ie they have the biological responses to the drugs for over 20,000 patients that is used that as a reference.

When you put an unknown sequence in from someone taking three drugs, you derive an estimate of virtual sensitivity to drugs by computer analysis. What is then reported is that based on the experience of the database the chances are there is a x-fold change in sensitivity for this patient.

The limitations to this approach are still being proven – it is a very nice concept but it needs to be proven. Limitations will be the size of the comparative database – the bigger it is the more powerful it is to actually make the right interpretation but new drugs on the market are unlikely to be represented in the database in the same way. Two years ago no-one in that database would have seen Kaletra and so it would be very difficult to get a virtual phenotype for Kaletra from that database. It requires the database to be updated all the time.

The capacity of the computer is also important. The analysis is actually done in bunches of mutations which are run into the machine and then analysed and often where it can’t get a good representation of a population within the computer it may then revert to a rules based interpretation because the information just doesn’t exist in the database.

Q: I was surprised that the virtual genotype does not compare your genotype to other exact matches of that group of mutations. It compared it to a different set of databases for each drug so that it would compare it to 8,000 people in the database that all had 3TC resistance … and 300 that might have ddI resistance. So you are not getting your exact genotype compared to someone else that also has your genotype.

A: No that’s right because I think if you made the computer focus down that hard you may only have ten patients to look at and the feeling was that isn’t enough to give you confident results.

Q: But would it not be useful to know what happened in those ten patients.

A: It may well be but we are talking about infomatics and systems that have to be developed to meet multiple needs which is why I think they have had to go down that pathway. I always think that is a very good point because you put in information about your patient like the drugs they are taking – now the database may have 16,000 patients in it may only find 100 patients to compare with and they actually put the number on it. They say this data and results have been created on the basis of, say, 100 patients who are quite close. It has limitations definitely and of course it all depends on how that data was built because if there was a trial of 1000 80-year old men the results may be different from a population of 20 year olds – so there could be skewed data in there.

Q: In Edinburgh most people want the results of their resistance test quite quickly and they come back in six days – at least this is fast.

A: Is that including the sequencing? One of the problems with the actual phenotype is the amount of time that it takes for it to come back.

Resistance Guidelines – when to use tests

There are a number of guidelines groups looking at how to use resistance testing – International Aids Society in the USA, Euro guidelines and BHIVA guidelines and I am involved in all of these to some degree.

This is partially scientific and partly driven by political pressure and they produce consensus recommendations for different groups.

For primary HIV infection: most guidelines say if early treatment is a local policy then you
should have minimum delay in treatment but they would recommend resistance testing at the same time as you start treatment and then you can make a modification of that treatment if within days of getting the result. We turn these around these tests very quickly because they are very important, and within three or four days of getting the result you can find whether someone has unfortunately acquired a drug resistance virus and therapy needs to be modified.

For chronic patients: in people who have not started treatment, the recommendation for most of these was ‘to consider’. I’ve always thought that resistance testing should be recommended for everyone before they start treatment – and these committees are now coming around to this point of view.

This is why this in bold (in slide 52). There is a tendency to move more to recommend it than just consider it. The consideration should include the background prevalence of resistance in any specific community, so if you have a background prevalence of10 % resistance you may say then we must test all our naive patients before we start their therapy.

My feeling always has been having decided to take drugs, the person’s first therapy is a major landmark in their lives. I always think that the response of that first therapy is very important, and to have a failure for a stupid reason like resistance that we could have tested for is devastating for the patient. So I’ve always said I think we should recommend testing people at baseline. I always think of myself when I am treating people and I think if I were starting antiretrovirals for the first time what would I want to do… I would want a resistance test before I start because it is a stupid reason for failing and failing is hard work for everybody but specifically for the patient.

Q: … at the beginning of your talk about viral load tests you were saying about individualising treatment… but even if the prevalence in the local area is low that doesn’t matter if the one particular person that you are seeing has resistance …

A: Absolutely right. This comes back to the individual – and that is why I always say what

would I like. But what you have to remember about these panels/committees is that they aren’t only driven by good science and good medicine, they are driven by good politics. There is always someone who is driving the politics saying, ‘you can’t do that… we can’t even afford to test the people who are on second and third therapy so how can you ever justify doing it for people who have never had therapy?’ Well I can justify it and I generally do what I want to do in my own little domain!

Q: So things are different in theory and in practice?

A: Of course, but isn’t life like that? People say there are good and bad doctors and people generally tend to find their way to the people they like to have their care with. Equally that may be little to do with care it may be to do with personality. There may be patients who like people shouting at them, there may be people who like people to be gentle with them. It is all taste in the end but when you come down to guidelines and rules you lose all that and you have to kind of do end up with publishable recommendations.

For second and third treatments: obviously resistance measuring is recommended in some form here. I regard each one of these stages in a treatment history as a therapeutic crossroads. We believe now in saving samples at each of these points for patients to use in the future. Even if we don’t test them we know that we have samples archived and we can go back to them at a later date to find what viruses may have accumulated in that patient. It becomes more important when you get towards the salvage stage.

For treatment in pregnancy: It is recommended in mums prior to starting treatment and who have detectable virus – but all these tests need a certain viral load copy to work (usually over 500 copies/ml).

Q: What is the cut off now?

A: We will try with patients down to 400 – 500 copies. You can increase the volume and try even harder but then you run into problems with subpopulations. Everyone is trying to improve the sensitivity of the tests but if you speak to the commercial boys they’ll say a thousand and probably in what we produce we say a thousand copies minimum.

Q: So what tests are being used when you see studies that look at resistance in people who are undetectable below 50? Are they just very finely tuned?

A: Yes, it is kind of specific hands on research where you might pull out single viral clones. PCR goes up and it is often very different technology. The issue if you go down to very low copy numbers is that because of the PCR technology what you can end up doing is just puling out one virus which you just amplify and there is no shotgun effect. You are not dealing with a population of viruses in that patient, you are pulling one out and that is useless because that patient has a whole population of viruses and if you just pull one out somewhere and say that is not resistant it doesn’t mean a thing. We actually have ways of looking at sequences to make sure we are looking at mixtures. If ever I see what looks like a clonal representation where there are no mixtures I would be very worried about reporting that because we may have amplified one virus where you are working at low copy numbers. That is not representative of what is happening in the patient.

Q: How about in a case where someone has been undetectable for four years, five years – what is their risk of resistance?

A: In theory yes but we are limited by the ability to amplify the whole population of viruses and give you a good consensus result of the sequence. We are all very interested in what is happening both in terms of viral load below the conventional cut off of 50 because we would like to know if there is further efficacy we could be finding down to three copies and equally what is happening in resistance. What we do know in studies that are being done in tissues it takes much longer for resistance to occur. It is always a function of replication rate. The more the virus multiplies the more you will evolve resistance but we know people even who are less than 50 do still tend to be replicating at a very low rate and producing new mutations at a very low rate, over years rather than months, but it is still going on and that is important to remember. We don’t know what is going on at these very low levels but we are now looking in an experimental way at that area.

Q: I know that you think that if someone has ongoing replication of between 50 and 500 it is inevitable that that combination will develop resistance and that it will eventually fail and rebound further but on that basis you would recommend changing treatment even when you have those low rebounds?

A: No I wouldn’t necessarily, it depends on the combination. Some patients have maintained 500 copies for a number of years, although this isn’t the pattern that you would expect. In biological terms some sort of equilibrium has taken place within the person, between the viruses that are growing, the drugs that are present and the whole immune response or the person. So you end up with some patients trickling along at 500 for years – and that may be a very happy state to stay in. Those decisions are usually made by different physicians and patients and it will vary from physician to physician as to what they want to do. We still have a very small number of patients who I see who are virologically stable on two drug therapy.

Q: …I have never worked out why resistant strains are actually less likely to occur in the wild type strain or less likely to survive?

A: By and large they are – it’s a trade off and when the virus mutates to grow or work in the presence of the drug, its trade off is that the efficiency of the enzyme gets less so actually in functional terms, what we might call fitness, it is less fit.

Q: They are at a disadvantage?

A: That’s right especially in minority populations. The minority virus may only have a few mutations but in terms of competing in the absence of drug wild-type majority virus have got more muscle so they will grow better.

Q: Minority viruses could become more dangerous if there was a change in treatment?

A: Absolutely. So sometimes the question is should we change at all because if we are going along at 500 copies for a number of years, you may stick with that – but you take into account all the other measures, the CD4 count, how many drug changes you’ve had, what other options are available to you and so on.

Q: Is there any evidence that per log increase in viral load and speed of resistance, mutation acquisition occurs faster i.e. form 50 to 500 or 500 to 5,000 etc

A: I don’t know of any evidence but if you ask me should it make a difference then yes, and if you
look at people who move from 50 to 500 who are evolving mutations they will go 50, 500, 5,000,
50,000 almost in a line that you can draw. But something different is happening in a group that stay at 500. That may be nothing to do with virus or drug but it may be a function of host factors that are doing something to the virus and while that is maintained – it depends on a whole host of things in relation to the patients care I think and the state of the disease they are at and what drugs they have available, but I don’t know of any evidence.

Q: Can you test to know whether even if you do have quite a large viral load you’ve may have a pretty unfit one?

A: No it hasn’t been used in clinical terms, it is an interesting question, and people are looking at
issues around fitness at the moment and technologies are being developed that are more slick than they had previously been. Previously they were very complicated and expensive that it wasn’t really being done. Certain approaches may make that a possible to look at in clinical terms now.

Q: My six year old has had a viral load reading in the last four years of about 2000
after a maximum of 20,000 – the last four years it has remained between 2,000 and 4,000 and it
hovers around. Originally it was 6,000,000. Due to other factors such as taste and getting a child to take liquid formualations, his treatment hasn’t changed. Is there a certain amount of fear of changing treatment because of this little one that is hiding down there, suddenly becoming stronger. The empirical evidence is that he is very well.

A: The first thing that I should say is that the virology of HIV in kids, I haven’t talked about it, but it is slightly different to that in adults and kids tend to start off with much higher numbers, probably because of the function of the immune system and other host factors. Viruses are allowed to grow much higher initially and under therapy a good response may not be less than 50, it may be less than 2,000 or 4,000 so you have to kind of shift your numbers for adults to kids. From the case you’ve suggested that may suggest the patient is being found stable at what is quite a satisfactory level. You also have to consider the drug options that may be available because they are less than they are in adults because the drugs may not be formulated or acceptable at the moment for children. So they are things that need to be weighed up.

Q: How reliable are resistance tests, and how much faith we can have in them? One of the things that struck me most was a report at Barcelona (from the GUESS Study) where they had put the same blood test out to all sorts of European centres who had come back with completely different sets of interpretations.

A: Yes well that will vary with certain systems, there’s more finesse developing in certain areas.

Q: Isn’t it more standardised now?

A: It gets more and more standardised as time goes on but if you are looking at guidelines and the panels who are looking, they can only look at research as it develops. There are different systems and one that BGI do is they give an interpretation of what a mutation may mean to drug efficacy and then also power it saying this data is from a randomised control trial – good strong evidence – or it might take it all the way down to case reports at a conference and what you would always be looking for as you re-review data from conference after conference. You look for the original findings to be powered up and then you may increase the power of it. So it is quite important when you look at the result and the interpretation to also see how a study is powered .

Q: GUESS was asking the experts to predict the phenotype from the genotype…

A: It is not easy and when I sit down to do it I like all the information I can get. You can’t just look at a sequence and say, ‘it is this, this and this so let’s give the patient that’, that’s not doing it at all. Talking to the doctor is usually the best way to do it because you want to know what the CD4 count is, how advanced the patient is, how many other therapies you actually have available anyway, how many drugs the individual is intolerant to and so on and then you would discuss and give advice on the possible options available. But discussing it is probably the most important part, that’s what we’ve always done in medicine, you do ward rounds, you don’t just write didactically on a piece of paper – you have this, this and this, therefore use these drugs – the physician would just look and say, ‘that’s crap, my patient has had this already and the mutations have disappeared’ or, ‘they are intolerant to this’, you actually need to discuss it.

Q: How many experts would you say there are in the UK?

A: I don’t keep abreast of it so I don’t know….

Q: What advice can you give to colleagues who are trying to prescribe these medications in a resource poor setting where there are no such facilities?

A: It is a major problem really because there are different views on this. We were treating patients when there was nothing to monitor therapy with except a CD4 count at one point in time and you have to make decisions about how you want to go forward. Do you want to take these therapies into resource poor settings and give them empirically knowing you will be having overall some benefit to those patients even without monitoring? Our view is that we are trying to develop low cost molecular technologies to try and support that activity in those settings you describe.

B and non-B clade viruses

Another problem in these settings, and I haven’t talked too much about it, is a vulnerability with PCR technology. Non-B subtypes were originally very much focussed on the African continent but are now found all over the world, because of the nature of movements of people. In the UK we have representations of every subtype of virus and once those viruses are in a community, if they are both inside one cell together they also recombine. They start sharing their genetic information with one another and so you get recombinant viruses. So we are finding new strains of viruses, or new subtypes of viruses, in our patients, so 20 % of our patients that we look after at a North London hospital are infected with non B viruses.

All PCR-based molecular tests have special primers or targets to make the PCR work. All of them were developed in the middle to late 80s using laboratory strains of subtype B virus, so all the kits we originally got were all for subtype B virus. There have been some difficulties – for example, one kit suddenly stopped working for non B viruses. Now those companies made adjustments and evaluations and altered them but because the virus is multiplying and evolving day by day it is not just an issue of saying, ‘we’ve corrected that kit now, it’s okay’. We need to monitor viruses in all our patients in the UK, in Europe, around the world continuously to watch out for evolution of viruses which could be undervalued or undetectable using molecular assays.

So currently at this point in time we have some patients who we cannot offer management to in terms of resistance measurements because we cannot amplify their viruses. So there are, even in this country, patients that cannot have a resistance test – they cannot have a sequence done because we can’t amplify their viruses.

Translate that to the countries you describe. I believe we should have some ways of monitoring. At Barcelona there was talk of stepping back eight years and using a p-24 test. – and there may be ways of using that to monitor whether treatment is working r when it is needed. Certainly we’ve demonstrated in patients where p-24 is detectable you get a good parallel result between viral load level and p-24 level but we also know that p-24 isn’t detectable in a large proportion of patients at a certain stage of the disease. The alternative is to use low tech approaches which may give you half log measures of viral load, to the nearest half log, and there are ways of doing that more cheaply and that may be a better way of monitoring. Equally there are CD4 counts and some of our colleagues, are working now in South Africa at developing a very low cost CD4 count and that also would be necessary to support patient care in those settings.

I think that we need to look at ways of translating this technology if we are going to use therapy in those settings. I know already, maybe not in Africa but say in Russia, I know that drugs have been provided and offered on a one or two drug basis. In the last few years of taking the drugs to those countries patients have rather taken one drug or two drugs rather than no drug and that is not ideal.

Maybe if we are looking at ways of attacking the 90 % of the epidemic which isn’t even benefiting from any of this at the moment, that is where we should be focusing lots of effort. It is sometimes very difficult to see, it is hard to do, there’s a lot of money being thrown at it at the moment – and a lot of people working on it – I just hope that at the grass roots level it is bringing the best benefit to the most patients and not becoming an academic exercise for people. We’ll have to see how that works out.

Clive Loveday

Aug 2002
UK-CAB / i-Base