- Email: [email protected]
Recommended procedures for the diagnosis of persistent active herpesvirus infections
Journal of Virological Methods, 21 (1988) 291-300 Elsevier
291
JVM 00783
Recommended procedures for the diagnosis of persistent active herpesvirus infections Gary R. Pearson Department of Microbiology,
Georgetown
University Medical School, Washington, U.S.A.
Summary While reliable tests are available to diagnose acute herpesvirus infections, this does not apply equally well to the identification of chronic active or recurrent infections. Common serology in most cases is not very reliable, unless the antibody response is carefully dissected to determine the response to some early antigens related to viral replication, such as against EA complex in Epstein-Barr virus infections. There are a number of techniques that hold some promise for the future: antigen demonstration by monoclonal antibodies such as in the shell vial assay or in tissue culture cells exposed to body fluids; in situ hybridization for viral DNA and polymerase chain reaction; and antigen assays by combined blottingimonoclonal antibody techniques. In developing meaningful parameters for defining persistent active or reactivated herpesvirus infections, techniques for quantitating virus and/or antigen should be helpful. Herpesvirus infections; Epstein-Barr virus; Acute infection; fection; Reactivated infection; Serology; Virus isolation
Persistent
Gerhard Krueger has asked me to summarize what has been meeting in regards to the development of assays for identification
Correspondence to: G.R. Pearson, School, Washington, DC U.S.A.
Department
of Microbiology,
Georgetown
active
in-
presented at this of active or per-
University
Medical
Editor’s comments: The following article and excerpts of the discussion have been transcribed from a tape recording taken during the workshop. Its comments appeared to us important enough to be published at this place. 0166-0934/88/$03.50
0
1988 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
292
sistent viral infections and to come up with a set of recommendations. Obviously, I think this we all agree will be difficult to do. I will try to come up with some more general comments in regard to these questions. I will open my comments by saying that in my opinion I do not think there has been a great deal of progress in the development of the tests for identifying particularly persistent active or recwrent viral infections. It is clear that we have made some progress in the development of new diagnostic tests for the diagnosis of primary infections. Progress certainly has been made, particularly with EBV and CMV, in the development of more sophisticated assays for clinical application, and also for the elimination of factors that give false positive results, such as, for example, elimination of rheumatoid factors, which is always a problem in the diagnosis of primary EBV infection. This is particularly true in diagnosing EBV and CMV. I think it is fairly clear that much more work is needed in regards to HBLV. as far as developing tests for the diagnosis of infections associated with this particular virus. This will require some fairly systematic studies which will be dependent on the identification of proteins associated with this virus that are produced in the infected cell, and on the identification of the immune response against these antigens before one can in fact really develop reliable tests for diagnosing HBLV infections. I think it’s fairly clear from this meeting with the amount of activity that is now ongoing on HBLV in a variety of different laboratories, that there will be rapid progress over the next year in developing tests for identifying HBLV infections and hopefully for identifying the diseases that may be associated with infections by this virus. Now, in regard to persistent or active infections, clearly more work is needed in these areas. Serology in most cases is really not very reliable or dependable in regards to making a diagnosis of a reactivated infection. The one possible exception is EBV, and I will illustrate this a little more later on. The most reliable and dependable assays for identifying active persistent and reactivated infections appear to be isolation assays. I think you have heard from a number of people at this meeting about new approaches for measuring or identifying viral shedding in the saliva or the urine, which would be indicative of persistent or active viral infections and which can be used from the diagnostic point of view. There are probably three major approaches for attempting to identify active infections by the herpesvirus. One, as we mentioned. is the serological approach, which requires the identification of specific viral protein produced during the viral replication. As I already mentioned, in most cases serology is not particularly good for identifying reactivated infection. If you are fortunate and have periodic serum samples of patients. one in fact can look for increase of antibody titers in these periodic serum samples which would correlate with the development of the clinical symptoms. However, this is not always the case. I think what one really needs to do in the serological assays to develop reliable or dependable assays for diagnosis of recurrent infection, is to dissect out the immune response against the viral antigens as has been done with EBV. It would be particularly important for all the herpesviruses to identify those antigens like the early antigens that are produced only during an active viral replication and the immune response against these antigens could be monitored as an indicator of active viral infection. Again I will il-
293
lustrate this with EBV. However, there are precautions that one has to take even under these circumstances. It is fairly clear that there are many people, for example in EBV infections, that have antibodies persistently against these early antigen complexes and have no particular active disease, so that one certainly has to relate serology to clinical symptoms before drawing any conclusions as to the role of the virus in the induction of the disease symptoms. There is no absolute, as far as I can tell, in using serology for the diagnosis of reactivated diseases. The second approach which has been used most consistently is virus isolation. That is, attempting to detect infectious virus in body fluids, usually saliva or urine. Now, in most cases it is not particularly meaningful just to be able to isolate virus. We know, in EBV infections for example, that most of us who have been exposed to the virus intermittently should shed virus in the saliva. So the fact that just finding virus in the saliva certainly does not mean anything. If you can show increases in the virus concentration in the saliva in a period of time, this in fact could be meaningful to suspect that EBV may be involved in some type of persistent infection. What we lack, I think, in the virus isolation area are good methods for quantitating the virus. The methods that have been presented at this meeting, certainly with CMV and EBV, are reliable for detecting the virus, but you really need to be able to quantitate it. I think this one area, that needs a great deal of more work, is development of reliable quantitative assays for measuring virus in different body fluids and try to relate this to disease symptoms. The third approach that is being used for trying to identify active infections is the direct detection of virus in circulating cells looking either for antigen or for viral DNA. Dr. Ablashi presented some data using monoclonal antibodies in people with immunodeficiency and using these antibodies to try in fact identifying antigens that may be expressed in increasing numbers of cells in the circulation, using this as an indicator for a persistent active infection. Quite exciting is the polymerase chain reaction presented by Dr. Bu~hbinder, and its potential application to this particular question. That is a very exciting technique and can provide very meaningful data in relationship to the detection of virus in circulating cells or in diseased tissue and in fact then to the diagnosis of those particular infections. I suspect that we will see a great deal more of that application over the next few years. In regards to tests for detection of cell associated virus, we have heard a great deal at this meeting particularly concerning the CMV on different approaches for detecting infectious virus in the urine, and on the clinical significance of these fmdings. The approaches that are taken have been the direct culture of the specimens, which has the disadvantage in that it takes a great period of time in many cases before one can say that a culture is positive or negative; in many cases it takes so long, that it’s really not meaningful clinically, because the clinician has to move to some sort of treatment regime before he gets the data. To overcome this, new assays have been developed with CMV and also, I believe, being developed with the herpes simplex virus that uses monoclonal antibodies in the shell vial assay or for antigen detection in virus-exposed cells (culture cells exposed to saliva or urine in suspected virus shedding). This is certainly a very promising rapid assay for the
294
detection of virus and for finally making the diagnosis. I just mentioned about the antibody status in circulating cells for the expression of replicating antigens, which can be used particularly with the development of monoclonal antibodies and also with the nucleic acid hybridization approaches looking for viral DNA or the polymerase chain reaction. None of these assays, however, is particularly good for quantitating virus; simply detection of virus is not adequate in helping the clinicians in the diagnosis of a disease. What one really has to do is to attempt to develop more quantitative rapid assays for the detection of virus that is being shed in the body fluids. One approach that we are working on in our laboratory with Dr. Luka is to develop antigen assay by a blotting method that can use monoclonal antibodies and where one could try to relate protein concentration with infectious virus; that one can develop a rapid blotting assay where one can take a specimen, blot it, detect the virus with the monoclonal antibody and then measure the different sensitivities by using different dilutions, relate them to infectious virus particles. make a standard curve and through the standard curve. could possibly get a rough estimate of the virus that may be in the circulating fluids. This has supposedly some potential in the future as rapid approach for quantitating virus in the circulation and in the body fluids. This could be used for all these herpesviruses. It is very clear that we do need a lot of work in this area before we will have meaningful assays for active persistent or reactivated infections. Now, in regards to the potentially useful serology: again, as I mentioned from the point of view of reactivated infections, serological assays are not very dependable. But the one that has been studied the most and is probably the most dependable and potentially the most meaningful is what has been done in EBV. This is because of all the work that has been done over the years in defining the various events during the virus replication cycle, defining those antigens that are important for the expression of the virus, the immune response against these antigens, and then the demonstration that in fact the immune responses against some of these antigens are meaningful in relationship to active infections. Basically, what one sees during the acute stage of EBV infection is an IgM and an IgG response against the viral capsid antigens which come up and then persist for life. They may remain stable at the peak titer or slightly drop off, but essentially the IgG titer will remain stable for life and will be your indicator that the patient was exposed to the virus. The IgM titer, as you know, will be transient, come in the acute phase and then disappearing in the convalescense. If you get the serum samples at the right time. you can look for IgM antibodies as an indicator for a primary infection. Now in addition, in primary infection in many but not all individuals that get infected with EBV, one does get an antibody response against this early antigen complex. This appears to be a reliable marker, a reasonable marker for the presence of an active infection. Following the primary infection in the classical situation one does get an antibody response against these early antigens; these antibodies will come up and will then disappear as the disease subsides, so that it seems to be a marker for active infection. Now this is not an absolute and does not happen in all situations. but one can monitor in many cases what we think is an active infection by follow-
ing this particular antibody. This is also suggested by a number of studies in patients with Burkitt’s lymphomas and nasopharyngeal carcinomas; in fact the antibody response against this complex has been reported to be diagnostic or prognostic in patients with these diseases. For example, antibodies against the early antigen complex are generally present in very high levels in patients with Burkitt’s lymphoma and nasopharyngeal carcinoma at the time they present with the disease. It has been reported in the literature that when patients are treated successfully these antibodies will drop. and in many cases will disappear indicating the good prognosis. They will reappear before tumor develops. This indicates that in these patients one in fact does have active viral replication and that the antibody response against these replication antigens appears to be of significance to the development and progression of the cancer. The reason for this is really not clear because we are not quite sure what persistent viral infections mean in regards to the persistence of a cancer. But the fact that antibodies do fluctuate make it a very nice prognostic tool to follow up these patients. The neutralizing antibody response parallels pretty much to what you see in the VCA response. Antibodies do come up during acute phase of infection and then essentially will persist for life. So again these really are not meaningful in regard to reactivated or active infections but will again indicate a past infection. The anti-EBNA response is delayed and usually does not come up until the convalescent phase and therefore can also be used for diagnosing primary infections but not necessarily reactivated infections. The APCC 1 will not go into details, but it falls very similar to the antibody response against EBNA. Again the key is the persisting antibodies against the early antigen complex; this is our best indicator for an active infection. If antibodies persist against EA, one suspects that there in fact is an active infection or persistent infection still ongoing in that individual. If antibodies in the ideal situation would in fact go up and then disappear and then re-occur with the clinical symptoms, this of course would be extremely meaningful from the point of view that EBV has been involved or associated with the clinical syndrome. These are certainly the type of assays that have been used to try to relate EBV to the chronic fatigue syndrome (CFS). As we heard from Dr. Straus, this probably has not been as meaningful for a relationship of EBV and CFS as one has hoped. But this is what one does as far as using serology for this particular virus. I spent some time on EBV since it is so well disected and the immune response has been so well dissected against the various proteins. I think that we need this type of dissection with the other herpesviruses. I think this is true with the CMV, herpes simplex and certainly HBLV, before one is going to make significant progress to develop assays, clinical assays that can be used to diagnose persistent or reactivated infections; these constitute an extremely difficult problem in the clinics and really requires immediate attention. So basically in this somewhat brief presentation of our current stand in regards to the assays. I think we again go back and conclude: we have made progress. and there are really quite good assays with many of the viruses, in diagnosis of the primary infection. It certainly requires much more attention to be placed on assays that might be used reliably for reactivated infections in the future. It is fairly clear
that many of the syndromes that we will be running into, that we are running into now, like CFS, potentially are related to reactivated infections in immunode~~ient populations. It is necessary, however. to define useful parameters either serologically or to quantitate virus that can be used in a clinical situation for diagnosing this particular type of infection.
Discussion ~~~~~2~: There are a couple of things on EA and on EBNA that I would like to comment on. You eluded to the part that EAD is not so reliable for diagnosis of persistent active infection as it is for acute infection because there are good reports now that EAD may persist at reasonably respectable titers in apparently totally healthy people. EAD one can find at titers of 40 or even higher persisting in ohviously healthy people. The other question is, do you find certain EBNAlIEBNA2 profiles of any importance in persistent infection’? Pearsoit: In regards to EAD I think you stated it very clearly: it’s not an all ot none situation. It’s not that you get an active infection and the disease with elevated antibodies against EAR and EAD which disappear in a 100% of the cases as the disease subsides. As you pointed out, there are many of us including myself who walk around with antibodies to some of the early antigen complexes with no apparent indication of some type of active infection. One has to look at it in the context of the clinical symptoms. If you have increases of antibody titers or if you have periodical samples which you can follow and see significant increases of the antibodies even in those individuals that have persistent levels, this is probably meaningful. but just the presence of EA does not mean anything necessarily. Second, the immune response against EBNA for a primary infection has been defined very nicely by the Henles and by Steve: you apparently get a transient EBNA antibody response initially against EBNA-2 which disappears and then EBNA-1 comes up and persists: this is the usual kinetics of the antibody response during a primary infection. In people with immune deficiency there is some indication in papers both of the Henles and of George Miller that these individuals show persisting levels of antibodies against EBNA-2 but don’t really show much antibody against EBNA1. So the ratios switch, while normal individuals after the primary infection the EBNA-UEBNA-2 ratio is high, in persons with immune deficiency have more antibodies against EBNA-2 instead of EBNA-1 and the ratio inverts. There have been more studies done on this, and initially it looked like the EBNA-2 monitoring might be very meaningful from the point of view of the chronic fatigue syndrome. We then did a fair amount of work in our laboratory monitoring both EBNA-2 and the ratio, We also had some hints that it might be true for people with immune deficiency: they did in fact seem to have antibodies against EBNA-2 instead of EBNA-I more frequently. However, then we went a step further and did a controlled blind study with Dr. Straus. He provided us with an extensive series of sera from patients with chronic fatigue syndrome and with other disorders. where we
297
ran anti-EBNA-1 and EBNA-2 titers and tried to relate the data to the disease. That really did not come out well. The ratio wasn’t dependable and you could not predict who had the immune deficiency, who had the potentially acquired chronic fatigue syndrome and who was normal. So my feeling is that the EBNA-1, EBNA2 story is not dependable with regards to reactivated infection or persistent infection. Ablushi: One of the questions I have is on the terminology of chronic fatigue syndrome (CFS) and one is on virus isolation. In all reports I have been looking through, the terminology of CFS is so vague with different names given to it. Is the terminology in the recent paper by Holmes the one we are going to go by or are there any differences? Second, in all these cases being reported, I can not find statements on the frequency of EBV isolation. Dr. Pagano, I think, presented one paper, and after that I have not seen any reports on virus isolation. Straus: Let me answer the second question first, that’s easier. We have done some studies on virus isolation, and in a series of patients found rates of recovery of EBV in the saliva to be in the neighborhood of about 20-30% of samples. In our ageand sex-matched controls it was about 15%. I.have not published that data, because I don’t think anyone has looked adequately at rates of shedding of Adenovirus in those patients, at rates of shedding of HSV and of CMV which certainly should be done in comparison. We have not.recovered many of those other viruses @om our patients. I think, however, what the syndrome is telling us is, that there are immune abnormalities which could lead to serological and perhaps even virological evidence of active viral infection without either of those things being primary to the disease itself. So I don’t know if the rates of recovery are going to be helpful. Also, rates of recovery of viruses like EBV are so variable in different laboratories, and if you believe Alan Rickinson and look hard enough, you can recover virus from 100% seropositive people. In terms of the nomenclature there is a lot of controversy in the report that Gary Holmes and a number of us have put together in last month’s Annals of Internal Medicine for case definition. The purpose of that case definition of CFS is that people who write about it can at least in some way compare the same types of patients from a clinical standpoint. Many people at this meeting have talked about patients with severe progressive chronic disease. To my mind, we need to distinguish among these things, that people who have the types of illnesses that Prof. Krueger just showed should not be lumped together under the chronic fatigue syndrome. First of all, it makes people who just have fatigue and some other kinds of constitutional complaints much more frightened, what they have may be a severe progressive disease, and in the others may minimize the impact of the type of illness that Prof. Krueger showed. I don’t actually care what you call these things as long as you don’t blur the distinction between them. Komaroff: I do agree with what Steve said. I think publication of a working case definition is a useful step, but you have to be concerned about the premature sanc-
ti~cation of any classi~cation scheme. I think what we should be doing are two things: when we report in the future on patients vvith CFS we should stratify our report into those that fully meet this new working case definition, and then describe the others who we may also choose to study and report even though they don’t fully meet the working case definition. because we can blind ourselves and exclude from the study valuable patients who might fall outside the case de~nition. I think. we should avoid doing that; we should simply carefully stratify patients into at least these two groups. The other thing we should be doing which may he more important than coalescing on a working case definition, is collecting the data required by that definition in a consistent way, for exampie depression. When we include or exclude patients from the working case definition on the basis of depression we should be defining depression in a consistent way. We should he asking about elements of the history in a consistent way. because much of the elements of the working case definition are themselves quite fuzzy. We can delude ourselves that we are describing the same patients when in fact we are collecting the essential data in quite variable ways. ~~~~~~~~~~~~: With regards to viralreactivation, couldn’t sitive cells in the circulation’! It this a possibility? Peurson: I think it’s a possibility. well. It has been tried in infectious mal individuals. it’s hard to find looking in patients with infectious positive cells. Again, I don’t think let Steve comment on it.
one look for EBNA-po-
I don’t think it has been really worked out very mononucleosis for example. If you Iook at norEBNA-positive cells, and there has been some mononucleosis for increased numbers of EBNAthis has been a real reliable method. but I will
Straus: Actually. there is some data on that yet. Giovanna Tosato in her group has studied for the past decade precursor frequency ratios, I suppose such as EBNA staining. They had looked at the proportion of circulating B-cells that will exhibit outgrow in serial dilutions in microtiter assays in normal individuals, who are EBV seropositive long after the resolution after acute infection: it‘s about l-10/10” circulating lymphocytes. There is a publication in the New England Journal of Medicine with Giovanna Tosato about a year and a half ago in which EBNA was shown in 100 to 500 cells per million cells. We actually looked with Giovanna at precursor frequency ratios in our patients with CFS as well as some of the patients with severe chronic EBV infections that I talked about shortly. Those are extremely variable, but there is a log full difference in our patients with CFS and the patients with these severe infections. These were relatively crude assays and I don’t think they are telling us enough about the activity of the virus. I might suggest that quant~tation of virus and saliva titer might be more meaningful. but in a dot&fat assay and serial dilutions there is certainly a correlation in the AIDS patients. The other thing is that if you detect EBV in tissues by in situ hybridization you are already detecting something that is a very rare process in the normat. You don’t see more than one cell in many fields, in EBV positive in normal individuals ex-
cept in an acute mononucleosis. I would encourage that.
That itself is in some ways a quantitative
test and
Pearson: I will make one other comment in regards to antigen and EBNA: looking at the replication antigens, in circulating cells of normal people that have been infected you usually can not find expression of EBV replication antigen. Looking in lymphocytes of people with immunodeficiency with the monoclonal antibody, you in general find an increase of cells that are expressing these antigens. Dharam reported this in a couple of situations and that might be very useful too, but again. it’s more developmental and needs some work to really relate the significance of this. I4’0lf I would like to make the foIlowing suggestion: when we isolate EBV from the saliva, I don’t see it terribly significant, because this might be just a physiological state that parotid cells may make a little bit more or less of the virus once at a time. Basically, we all can secrete it, we all synthesize it. Just counting EBNApositive cells in the periphery does not seem terribly significant in identifying cases like Dr. Krueger has presented. But what I would foresee is, to look for those sites in the human body which are probably capable of replicating EBV, but which are under control in the normal immunocompetent person, i.e. EBV-positive cells in those sites are eliminated. What sites are these, for instance? My suggestion is, to make touch preps from the side of the tongue where we know that cells are perfectly capable for making EBV. Such imprints from the side of the tongue are positive in AIDS patients, but negative in healthy indi~~iduals. Grifjth: I try to be a little bit more optimistic about the chance of quantitating virus, because some of the studies have already been done. Back in 1975 Stanley and colleagues published about virus titrations in CMV in urines from children who have been born with congenital infection. They showed that the ones who developed symptoms had at the time of birth 2 logs of the virus titers higher than those who didn’t develop symptoms. That’s quite a significant difference. Perhaps we shouldn’t be too pessimistic about trying to quantitate virus. What is needed, is an immunochemical or DNA method that’s more convenient than doing full tube titration on every particular patient; we need a rapid method for quantitating virus. Pearson: I agree, I think the EBV system I don’t assays are so complicated we really need are some have.
in the CMV system quantitation is quite meaningful. In think it has been worked out to the same point, and the that I don’t think we can say, it’s an absolute. But what more rapid assays for quantitating virus than all we now
Linde: I personally see one more aspect in this thing and that’s the story about pathogenesis. I mean, we all know that the majority of primary EBV infections are asymptomatic. Thus what makes an EBV infection, or CMV infection, or any other infection, what makes them symptomatic in some individuals and not in oth-
3w.l
ers? That question may not be answered by enumeration of virus or antibodies or things like that. but may go into lymphokines and many other things. I think this problem is so complicated that it needs a cooperation between immunologists, pathologists, virologists before we can answer anything of any of those questions really. Pearson:
I agree,
that’s a very good point.
291
JVM 00783
Recommended procedures for the diagnosis of persistent active herpesvirus infections Gary R. Pearson Department of Microbiology,
Georgetown
University Medical School, Washington, U.S.A.
Summary While reliable tests are available to diagnose acute herpesvirus infections, this does not apply equally well to the identification of chronic active or recurrent infections. Common serology in most cases is not very reliable, unless the antibody response is carefully dissected to determine the response to some early antigens related to viral replication, such as against EA complex in Epstein-Barr virus infections. There are a number of techniques that hold some promise for the future: antigen demonstration by monoclonal antibodies such as in the shell vial assay or in tissue culture cells exposed to body fluids; in situ hybridization for viral DNA and polymerase chain reaction; and antigen assays by combined blottingimonoclonal antibody techniques. In developing meaningful parameters for defining persistent active or reactivated herpesvirus infections, techniques for quantitating virus and/or antigen should be helpful. Herpesvirus infections; Epstein-Barr virus; Acute infection; fection; Reactivated infection; Serology; Virus isolation
Persistent
Gerhard Krueger has asked me to summarize what has been meeting in regards to the development of assays for identification
Correspondence to: G.R. Pearson, School, Washington, DC U.S.A.
Department
of Microbiology,
Georgetown
active
in-
presented at this of active or per-
University
Medical
Editor’s comments: The following article and excerpts of the discussion have been transcribed from a tape recording taken during the workshop. Its comments appeared to us important enough to be published at this place. 0166-0934/88/$03.50
0
1988 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
292
sistent viral infections and to come up with a set of recommendations. Obviously, I think this we all agree will be difficult to do. I will try to come up with some more general comments in regard to these questions. I will open my comments by saying that in my opinion I do not think there has been a great deal of progress in the development of the tests for identifying particularly persistent active or recwrent viral infections. It is clear that we have made some progress in the development of new diagnostic tests for the diagnosis of primary infections. Progress certainly has been made, particularly with EBV and CMV, in the development of more sophisticated assays for clinical application, and also for the elimination of factors that give false positive results, such as, for example, elimination of rheumatoid factors, which is always a problem in the diagnosis of primary EBV infection. This is particularly true in diagnosing EBV and CMV. I think it is fairly clear that much more work is needed in regards to HBLV. as far as developing tests for the diagnosis of infections associated with this particular virus. This will require some fairly systematic studies which will be dependent on the identification of proteins associated with this virus that are produced in the infected cell, and on the identification of the immune response against these antigens before one can in fact really develop reliable tests for diagnosing HBLV infections. I think it’s fairly clear from this meeting with the amount of activity that is now ongoing on HBLV in a variety of different laboratories, that there will be rapid progress over the next year in developing tests for identifying HBLV infections and hopefully for identifying the diseases that may be associated with infections by this virus. Now, in regard to persistent or active infections, clearly more work is needed in these areas. Serology in most cases is really not very reliable or dependable in regards to making a diagnosis of a reactivated infection. The one possible exception is EBV, and I will illustrate this a little more later on. The most reliable and dependable assays for identifying active persistent and reactivated infections appear to be isolation assays. I think you have heard from a number of people at this meeting about new approaches for measuring or identifying viral shedding in the saliva or the urine, which would be indicative of persistent or active viral infections and which can be used from the diagnostic point of view. There are probably three major approaches for attempting to identify active infections by the herpesvirus. One, as we mentioned. is the serological approach, which requires the identification of specific viral protein produced during the viral replication. As I already mentioned, in most cases serology is not particularly good for identifying reactivated infection. If you are fortunate and have periodic serum samples of patients. one in fact can look for increase of antibody titers in these periodic serum samples which would correlate with the development of the clinical symptoms. However, this is not always the case. I think what one really needs to do in the serological assays to develop reliable or dependable assays for diagnosis of recurrent infection, is to dissect out the immune response against the viral antigens as has been done with EBV. It would be particularly important for all the herpesviruses to identify those antigens like the early antigens that are produced only during an active viral replication and the immune response against these antigens could be monitored as an indicator of active viral infection. Again I will il-
293
lustrate this with EBV. However, there are precautions that one has to take even under these circumstances. It is fairly clear that there are many people, for example in EBV infections, that have antibodies persistently against these early antigen complexes and have no particular active disease, so that one certainly has to relate serology to clinical symptoms before drawing any conclusions as to the role of the virus in the induction of the disease symptoms. There is no absolute, as far as I can tell, in using serology for the diagnosis of reactivated diseases. The second approach which has been used most consistently is virus isolation. That is, attempting to detect infectious virus in body fluids, usually saliva or urine. Now, in most cases it is not particularly meaningful just to be able to isolate virus. We know, in EBV infections for example, that most of us who have been exposed to the virus intermittently should shed virus in the saliva. So the fact that just finding virus in the saliva certainly does not mean anything. If you can show increases in the virus concentration in the saliva in a period of time, this in fact could be meaningful to suspect that EBV may be involved in some type of persistent infection. What we lack, I think, in the virus isolation area are good methods for quantitating the virus. The methods that have been presented at this meeting, certainly with CMV and EBV, are reliable for detecting the virus, but you really need to be able to quantitate it. I think this one area, that needs a great deal of more work, is development of reliable quantitative assays for measuring virus in different body fluids and try to relate this to disease symptoms. The third approach that is being used for trying to identify active infections is the direct detection of virus in circulating cells looking either for antigen or for viral DNA. Dr. Ablashi presented some data using monoclonal antibodies in people with immunodeficiency and using these antibodies to try in fact identifying antigens that may be expressed in increasing numbers of cells in the circulation, using this as an indicator for a persistent active infection. Quite exciting is the polymerase chain reaction presented by Dr. Bu~hbinder, and its potential application to this particular question. That is a very exciting technique and can provide very meaningful data in relationship to the detection of virus in circulating cells or in diseased tissue and in fact then to the diagnosis of those particular infections. I suspect that we will see a great deal more of that application over the next few years. In regards to tests for detection of cell associated virus, we have heard a great deal at this meeting particularly concerning the CMV on different approaches for detecting infectious virus in the urine, and on the clinical significance of these fmdings. The approaches that are taken have been the direct culture of the specimens, which has the disadvantage in that it takes a great period of time in many cases before one can say that a culture is positive or negative; in many cases it takes so long, that it’s really not meaningful clinically, because the clinician has to move to some sort of treatment regime before he gets the data. To overcome this, new assays have been developed with CMV and also, I believe, being developed with the herpes simplex virus that uses monoclonal antibodies in the shell vial assay or for antigen detection in virus-exposed cells (culture cells exposed to saliva or urine in suspected virus shedding). This is certainly a very promising rapid assay for the
294
detection of virus and for finally making the diagnosis. I just mentioned about the antibody status in circulating cells for the expression of replicating antigens, which can be used particularly with the development of monoclonal antibodies and also with the nucleic acid hybridization approaches looking for viral DNA or the polymerase chain reaction. None of these assays, however, is particularly good for quantitating virus; simply detection of virus is not adequate in helping the clinicians in the diagnosis of a disease. What one really has to do is to attempt to develop more quantitative rapid assays for the detection of virus that is being shed in the body fluids. One approach that we are working on in our laboratory with Dr. Luka is to develop antigen assay by a blotting method that can use monoclonal antibodies and where one could try to relate protein concentration with infectious virus; that one can develop a rapid blotting assay where one can take a specimen, blot it, detect the virus with the monoclonal antibody and then measure the different sensitivities by using different dilutions, relate them to infectious virus particles. make a standard curve and through the standard curve. could possibly get a rough estimate of the virus that may be in the circulating fluids. This has supposedly some potential in the future as rapid approach for quantitating virus in the circulation and in the body fluids. This could be used for all these herpesviruses. It is very clear that we do need a lot of work in this area before we will have meaningful assays for active persistent or reactivated infections. Now, in regards to the potentially useful serology: again, as I mentioned from the point of view of reactivated infections, serological assays are not very dependable. But the one that has been studied the most and is probably the most dependable and potentially the most meaningful is what has been done in EBV. This is because of all the work that has been done over the years in defining the various events during the virus replication cycle, defining those antigens that are important for the expression of the virus, the immune response against these antigens, and then the demonstration that in fact the immune responses against some of these antigens are meaningful in relationship to active infections. Basically, what one sees during the acute stage of EBV infection is an IgM and an IgG response against the viral capsid antigens which come up and then persist for life. They may remain stable at the peak titer or slightly drop off, but essentially the IgG titer will remain stable for life and will be your indicator that the patient was exposed to the virus. The IgM titer, as you know, will be transient, come in the acute phase and then disappearing in the convalescense. If you get the serum samples at the right time. you can look for IgM antibodies as an indicator for a primary infection. Now in addition, in primary infection in many but not all individuals that get infected with EBV, one does get an antibody response against this early antigen complex. This appears to be a reliable marker, a reasonable marker for the presence of an active infection. Following the primary infection in the classical situation one does get an antibody response against these early antigens; these antibodies will come up and will then disappear as the disease subsides, so that it seems to be a marker for active infection. Now this is not an absolute and does not happen in all situations. but one can monitor in many cases what we think is an active infection by follow-
ing this particular antibody. This is also suggested by a number of studies in patients with Burkitt’s lymphomas and nasopharyngeal carcinomas; in fact the antibody response against this complex has been reported to be diagnostic or prognostic in patients with these diseases. For example, antibodies against the early antigen complex are generally present in very high levels in patients with Burkitt’s lymphoma and nasopharyngeal carcinoma at the time they present with the disease. It has been reported in the literature that when patients are treated successfully these antibodies will drop. and in many cases will disappear indicating the good prognosis. They will reappear before tumor develops. This indicates that in these patients one in fact does have active viral replication and that the antibody response against these replication antigens appears to be of significance to the development and progression of the cancer. The reason for this is really not clear because we are not quite sure what persistent viral infections mean in regards to the persistence of a cancer. But the fact that antibodies do fluctuate make it a very nice prognostic tool to follow up these patients. The neutralizing antibody response parallels pretty much to what you see in the VCA response. Antibodies do come up during acute phase of infection and then essentially will persist for life. So again these really are not meaningful in regard to reactivated or active infections but will again indicate a past infection. The anti-EBNA response is delayed and usually does not come up until the convalescent phase and therefore can also be used for diagnosing primary infections but not necessarily reactivated infections. The APCC 1 will not go into details, but it falls very similar to the antibody response against EBNA. Again the key is the persisting antibodies against the early antigen complex; this is our best indicator for an active infection. If antibodies persist against EA, one suspects that there in fact is an active infection or persistent infection still ongoing in that individual. If antibodies in the ideal situation would in fact go up and then disappear and then re-occur with the clinical symptoms, this of course would be extremely meaningful from the point of view that EBV has been involved or associated with the clinical syndrome. These are certainly the type of assays that have been used to try to relate EBV to the chronic fatigue syndrome (CFS). As we heard from Dr. Straus, this probably has not been as meaningful for a relationship of EBV and CFS as one has hoped. But this is what one does as far as using serology for this particular virus. I spent some time on EBV since it is so well disected and the immune response has been so well dissected against the various proteins. I think that we need this type of dissection with the other herpesviruses. I think this is true with the CMV, herpes simplex and certainly HBLV, before one is going to make significant progress to develop assays, clinical assays that can be used to diagnose persistent or reactivated infections; these constitute an extremely difficult problem in the clinics and really requires immediate attention. So basically in this somewhat brief presentation of our current stand in regards to the assays. I think we again go back and conclude: we have made progress. and there are really quite good assays with many of the viruses, in diagnosis of the primary infection. It certainly requires much more attention to be placed on assays that might be used reliably for reactivated infections in the future. It is fairly clear
that many of the syndromes that we will be running into, that we are running into now, like CFS, potentially are related to reactivated infections in immunode~~ient populations. It is necessary, however. to define useful parameters either serologically or to quantitate virus that can be used in a clinical situation for diagnosing this particular type of infection.
Discussion ~~~~~2~: There are a couple of things on EA and on EBNA that I would like to comment on. You eluded to the part that EAD is not so reliable for diagnosis of persistent active infection as it is for acute infection because there are good reports now that EAD may persist at reasonably respectable titers in apparently totally healthy people. EAD one can find at titers of 40 or even higher persisting in ohviously healthy people. The other question is, do you find certain EBNAlIEBNA2 profiles of any importance in persistent infection’? Pearsoit: In regards to EAD I think you stated it very clearly: it’s not an all ot none situation. It’s not that you get an active infection and the disease with elevated antibodies against EAR and EAD which disappear in a 100% of the cases as the disease subsides. As you pointed out, there are many of us including myself who walk around with antibodies to some of the early antigen complexes with no apparent indication of some type of active infection. One has to look at it in the context of the clinical symptoms. If you have increases of antibody titers or if you have periodical samples which you can follow and see significant increases of the antibodies even in those individuals that have persistent levels, this is probably meaningful. but just the presence of EA does not mean anything necessarily. Second, the immune response against EBNA for a primary infection has been defined very nicely by the Henles and by Steve: you apparently get a transient EBNA antibody response initially against EBNA-2 which disappears and then EBNA-1 comes up and persists: this is the usual kinetics of the antibody response during a primary infection. In people with immune deficiency there is some indication in papers both of the Henles and of George Miller that these individuals show persisting levels of antibodies against EBNA-2 but don’t really show much antibody against EBNA1. So the ratios switch, while normal individuals after the primary infection the EBNA-UEBNA-2 ratio is high, in persons with immune deficiency have more antibodies against EBNA-2 instead of EBNA-1 and the ratio inverts. There have been more studies done on this, and initially it looked like the EBNA-2 monitoring might be very meaningful from the point of view of the chronic fatigue syndrome. We then did a fair amount of work in our laboratory monitoring both EBNA-2 and the ratio, We also had some hints that it might be true for people with immune deficiency: they did in fact seem to have antibodies against EBNA-2 instead of EBNA-I more frequently. However, then we went a step further and did a controlled blind study with Dr. Straus. He provided us with an extensive series of sera from patients with chronic fatigue syndrome and with other disorders. where we
297
ran anti-EBNA-1 and EBNA-2 titers and tried to relate the data to the disease. That really did not come out well. The ratio wasn’t dependable and you could not predict who had the immune deficiency, who had the potentially acquired chronic fatigue syndrome and who was normal. So my feeling is that the EBNA-1, EBNA2 story is not dependable with regards to reactivated infection or persistent infection. Ablushi: One of the questions I have is on the terminology of chronic fatigue syndrome (CFS) and one is on virus isolation. In all reports I have been looking through, the terminology of CFS is so vague with different names given to it. Is the terminology in the recent paper by Holmes the one we are going to go by or are there any differences? Second, in all these cases being reported, I can not find statements on the frequency of EBV isolation. Dr. Pagano, I think, presented one paper, and after that I have not seen any reports on virus isolation. Straus: Let me answer the second question first, that’s easier. We have done some studies on virus isolation, and in a series of patients found rates of recovery of EBV in the saliva to be in the neighborhood of about 20-30% of samples. In our ageand sex-matched controls it was about 15%. I.have not published that data, because I don’t think anyone has looked adequately at rates of shedding of Adenovirus in those patients, at rates of shedding of HSV and of CMV which certainly should be done in comparison. We have not.recovered many of those other viruses @om our patients. I think, however, what the syndrome is telling us is, that there are immune abnormalities which could lead to serological and perhaps even virological evidence of active viral infection without either of those things being primary to the disease itself. So I don’t know if the rates of recovery are going to be helpful. Also, rates of recovery of viruses like EBV are so variable in different laboratories, and if you believe Alan Rickinson and look hard enough, you can recover virus from 100% seropositive people. In terms of the nomenclature there is a lot of controversy in the report that Gary Holmes and a number of us have put together in last month’s Annals of Internal Medicine for case definition. The purpose of that case definition of CFS is that people who write about it can at least in some way compare the same types of patients from a clinical standpoint. Many people at this meeting have talked about patients with severe progressive chronic disease. To my mind, we need to distinguish among these things, that people who have the types of illnesses that Prof. Krueger just showed should not be lumped together under the chronic fatigue syndrome. First of all, it makes people who just have fatigue and some other kinds of constitutional complaints much more frightened, what they have may be a severe progressive disease, and in the others may minimize the impact of the type of illness that Prof. Krueger showed. I don’t actually care what you call these things as long as you don’t blur the distinction between them. Komaroff: I do agree with what Steve said. I think publication of a working case definition is a useful step, but you have to be concerned about the premature sanc-
ti~cation of any classi~cation scheme. I think what we should be doing are two things: when we report in the future on patients vvith CFS we should stratify our report into those that fully meet this new working case definition, and then describe the others who we may also choose to study and report even though they don’t fully meet the working case definition. because we can blind ourselves and exclude from the study valuable patients who might fall outside the case de~nition. I think. we should avoid doing that; we should simply carefully stratify patients into at least these two groups. The other thing we should be doing which may he more important than coalescing on a working case definition, is collecting the data required by that definition in a consistent way, for exampie depression. When we include or exclude patients from the working case definition on the basis of depression we should be defining depression in a consistent way. We should he asking about elements of the history in a consistent way. because much of the elements of the working case definition are themselves quite fuzzy. We can delude ourselves that we are describing the same patients when in fact we are collecting the essential data in quite variable ways. ~~~~~~~~~~~~: With regards to viralreactivation, couldn’t sitive cells in the circulation’! It this a possibility? Peurson: I think it’s a possibility. well. It has been tried in infectious mal individuals. it’s hard to find looking in patients with infectious positive cells. Again, I don’t think let Steve comment on it.
one look for EBNA-po-
I don’t think it has been really worked out very mononucleosis for example. If you Iook at norEBNA-positive cells, and there has been some mononucleosis for increased numbers of EBNAthis has been a real reliable method. but I will
Straus: Actually. there is some data on that yet. Giovanna Tosato in her group has studied for the past decade precursor frequency ratios, I suppose such as EBNA staining. They had looked at the proportion of circulating B-cells that will exhibit outgrow in serial dilutions in microtiter assays in normal individuals, who are EBV seropositive long after the resolution after acute infection: it‘s about l-10/10” circulating lymphocytes. There is a publication in the New England Journal of Medicine with Giovanna Tosato about a year and a half ago in which EBNA was shown in 100 to 500 cells per million cells. We actually looked with Giovanna at precursor frequency ratios in our patients with CFS as well as some of the patients with severe chronic EBV infections that I talked about shortly. Those are extremely variable, but there is a log full difference in our patients with CFS and the patients with these severe infections. These were relatively crude assays and I don’t think they are telling us enough about the activity of the virus. I might suggest that quant~tation of virus and saliva titer might be more meaningful. but in a dot&fat assay and serial dilutions there is certainly a correlation in the AIDS patients. The other thing is that if you detect EBV in tissues by in situ hybridization you are already detecting something that is a very rare process in the normat. You don’t see more than one cell in many fields, in EBV positive in normal individuals ex-
cept in an acute mononucleosis. I would encourage that.
That itself is in some ways a quantitative
test and
Pearson: I will make one other comment in regards to antigen and EBNA: looking at the replication antigens, in circulating cells of normal people that have been infected you usually can not find expression of EBV replication antigen. Looking in lymphocytes of people with immunodeficiency with the monoclonal antibody, you in general find an increase of cells that are expressing these antigens. Dharam reported this in a couple of situations and that might be very useful too, but again. it’s more developmental and needs some work to really relate the significance of this. I4’0lf I would like to make the foIlowing suggestion: when we isolate EBV from the saliva, I don’t see it terribly significant, because this might be just a physiological state that parotid cells may make a little bit more or less of the virus once at a time. Basically, we all can secrete it, we all synthesize it. Just counting EBNApositive cells in the periphery does not seem terribly significant in identifying cases like Dr. Krueger has presented. But what I would foresee is, to look for those sites in the human body which are probably capable of replicating EBV, but which are under control in the normal immunocompetent person, i.e. EBV-positive cells in those sites are eliminated. What sites are these, for instance? My suggestion is, to make touch preps from the side of the tongue where we know that cells are perfectly capable for making EBV. Such imprints from the side of the tongue are positive in AIDS patients, but negative in healthy indi~~iduals. Grifjth: I try to be a little bit more optimistic about the chance of quantitating virus, because some of the studies have already been done. Back in 1975 Stanley and colleagues published about virus titrations in CMV in urines from children who have been born with congenital infection. They showed that the ones who developed symptoms had at the time of birth 2 logs of the virus titers higher than those who didn’t develop symptoms. That’s quite a significant difference. Perhaps we shouldn’t be too pessimistic about trying to quantitate virus. What is needed, is an immunochemical or DNA method that’s more convenient than doing full tube titration on every particular patient; we need a rapid method for quantitating virus. Pearson: I agree, I think the EBV system I don’t assays are so complicated we really need are some have.
in the CMV system quantitation is quite meaningful. In think it has been worked out to the same point, and the that I don’t think we can say, it’s an absolute. But what more rapid assays for quantitating virus than all we now
Linde: I personally see one more aspect in this thing and that’s the story about pathogenesis. I mean, we all know that the majority of primary EBV infections are asymptomatic. Thus what makes an EBV infection, or CMV infection, or any other infection, what makes them symptomatic in some individuals and not in oth-
3w.l
ers? That question may not be answered by enumeration of virus or antibodies or things like that. but may go into lymphokines and many other things. I think this problem is so complicated that it needs a cooperation between immunologists, pathologists, virologists before we can answer anything of any of those questions really. Pearson:
I agree,
that’s a very good point.