Introduction
It is now over 20 years since the first known HIV seroconversion in a haemophilic individual in the UK [1]. Between 1979 and 1985, when heat-treated concentrates were introduced to the UK, 1227 of the 6278 haemophilic individuals in the country (19.5%) became infected with HIV [2]. Because these men were infected so early in the epidemic, many died before the introduction of antiretroviral therapy (ART) and few have received highly active antiretroviral therapy (HAART). The majority of haemophilic men infected with HIV are chronically infected with hepatitis C virus (HCV) [3], which has implications both for their clinical prognosis and for their choice of and response to ART [4-6].
In 1989, we first described a cohort of HIV-infected haemophilic men treated at the Royal Free Hospital Haemophilia Centre (RFHHC) [7]. The earliest person to seroconvert in this cohort did so 19.5 years ago; therefore, this represents one of the longest prospectively followed cohorts of HIV seroconverters in the world. In this paper, we focus on the clinical status of these patients at April 1999 and on their uptake of and response to ART.
Patients and methods
Patients
The cohort of 111 haemophilic men, all initially registered at the RFHHC, seroconverted to HIV between 1979 and 1985 and have previously been described [7,8]. By April 1999, the cut-off date for this analysis, the patients had been followed for a median 16.9 years (range 8.0-19.5). Since the start of the study, 11 patients transferred their medical care to other centres. Vital status and information on AIDS status has been obtained through direct contact with these patients or their treating physicians.
The availability of stored serum samples enabled us to test samples for HIV seropositivity retrospectively and estimate seroconversion dates for each patient [7]. The patients ranged in age from 1.9 to 77.8 years (median 22.6) at the time of seroconversion. All patients had been exposed to HCV either before or at the time of infection with HIV.
Treatment
Patients were offered ART in line with the guidelines in place at the time. Since late 1995, patients have received HAART regimens including at least one protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI). The decision to use PI therapy in patients from this group is made cautiously because of the possible implications of liver complications in patients coinfected with HCV, and because of a number of reported bleeding episodes in haemophilic men receiving PI therapy [6,9].
Since 1988, patients with CD4 cell counts < 200 × 106 cells/l have been offered prophylaxis against Pneumocystis carinii pneumonia (PCP) and Candida spp. In addition, since 1991, patients have been offered prophylaxis against Mycobacterium avium intracellulare if their CD4 cell count fell to < 50 × 106 cells/l.
Laboratory methods
As part of a study on the natural history of viral load throughout HIV infection [8], HIV RNA levels were retrospectively measured on serum samples collected yearly after HIV-1 seroconversion using the Roche Amplicor HIV-1 Monitor v1.0 assay plus add-in non-B primers (range 400-750 000 copies/ml; Roche Diagnostic Systems, Branchburg, New Jersey, USA). Since 1996, HIV-1 RNA has been measured prospectively on fresh plasma samples using this assay and the ultrasensitive version of this assay (range 50-75 000 copies/ml). All HIV-1 RNA measurements are reported on the log10 scale.
Since 1982, lymphocyte subset analysis has been routinely performed on all fresh samples at the hospital, as described previously [8].
Statistical methods
Progression to clinically defined AIDS (a CD4 cell count < 200 × 106 cells/l was not considered AIDS defining) and survival were analysed using the Kaplan-Meier method. Patient follow-up was considered from the time of HIV seroconversion until the time of initial AIDS-defining event or death. Follow-up of patients who remained AIDS free and alive was right-censored on 30 April 1999. For some patients who had transferred their care elsewhere, information about the patient could only be obtained at some point prior to 30 April 1999; therefore, in these patients, follow-up was right-censored on the date on which information about their current vital/AIDS status had been received. Where information was not available on current AIDS status, or where the date of development of AIDS was not given, patient follow-up was right-censored 3 months after the individual's last visit to the RFHHC.
Patient follow-up was stratified into seven different calendar periods (prior to 1987, 1987-1988, 1989-1990, 1991-1992, 1993-1994, 1995-1996 and 1997 onwards). Clinical events occurring in each of these time periods were summed and divided by the total patient-years of follow-up in each period, to give an event rate for each calendar period. Confidence intervals (CI) for these rates were calculated using the exact Poisson distribution when the number of events in a stratum was less than 20, and the normal approximation to the Poisson distribution when the number of events was larger [10].
Comparisons of continuous measurements in patients currently under follow-up who had/had not received ART, were performed using the Mann-Whitney U test; comparisons of categorical measures were performed using the chi squared test, or Fisher's exact test where expected frequencies were small.
In order to summarize the information regarding changes in HIV RNA and CD4 cell count following treatment with HAART (defined for this study as any treatment combination of three or more drugs, including at least one PI or NNRTI), two sets of analyses were performed. First, the mean change in RNA level and CD4 cell count in each 3-month period following the start of HAART (1-3, 4-6, 7-9 and 10-12 months) was calculated for each patient. The median and 95% CI of the values at each time point were plotted. These analyses included all follow-up measurements irrespective of whether the patient had changed or discontinued therapy. Therefore, as a second analysis, we considered the time it took for the RNA level to fall to < 400 copies/ml in those individuals starting HAART with an RNA level > 400 copies/ml. Kaplan-Meier methods were used to summarize this information. Patient follow-up was right-censored if an individual changed their initial HAART regimen, at death or on 30 April 1999 if they had not had an RNA level < 400 copies/ml at that point.
All analyses were performed using the SAS software package [11].
Results
Progression to AIDS
By the end of April 1999, 57 patients (51.4%) had developed AIDS, a Kaplan-Meier progression rate of 57.0% by 19.5 years after seroconversion (95% CI 46.9-67.0) (Fig. 1), with a median time to AIDS diagnosis of 13.6 years. Eighteen patients (31.6% of those with AIDS) developed PCP as their initial AIDS-defining disease. Other common initial AIDS-defining events were oesophageal candidal infection (8; 14.0%), lymphoma (5; 8.8%), HIV encephalopathy (4; 7.0%), cryptosporidiosis, cytomegalovirus (CMV) disease, toxoplasmosis and mycobacterial infections (3 each; 5.3%). The pattern of subsequent events was broadly similar, although mycobacterial infections (eight subsequent events) and CMV infections (seven subsequent events) were much more frequently observed as a subsequent condition than as initial AIDS-defining events.
The rate of initial AIDS-defining diagnoses peaked in the period 1987-1988 (Table 1), with 1.03 events per 10 person-years of follow-up. The rate then dropped but remained relatively stable at between 0.47 and 0.77 events per 10 person-years of follow-up until the end of 1996. Since then, only one patient has developed an initial AIDS-defining illness, giving a rate of 0.13 events per 10 person-years of follow-up in the period 1997-1999.
Survival
By the end of April 1999, 65 patients (58.6%) had died, a Kaplan-Meier progression rate of 65.1% by 19.5 years after seroconversion (95% CI 52.7-77.4) (Fig. 1), with a median survival of 14.4 years. Death rates from 1987 onwards remained stable at between 0.56 and 1.00 per 10 person-years of follow-up (Table 1). Noticeably, there has been no drop in death rates since 1997; indeed the death rate in the period 1997-1999 is the second highest over all follow-up periods.
Until 1995, almost all deaths were in individuals with an AIDS diagnosis (43 of 50 deaths; 86.0%). However, since 1995, this proportion has dropped: only 8 of the 15 deaths occurring since 1995 (53.3%) have occurred in individuals who had a prior AIDS diagnosis. When we considered the actual cause of death in these individuals, the difference was even more striking. Of the 50 deaths occurring prior to 1995, 35 (70.0%) were directly from AIDS causes, five (10.0%) were caused by liver-related disease and 10 (20.0%) were from other apparently unrelated causes. In contrast, of the 15 deaths occurring since 1995, only seven (46.7%) were directly from AIDS causes, five (33.3%) were from liver-related causes and the remaining three (20.0%) were from `unrelated' causes (P = 0.09 for the two time periods, Fisher's exact test).
Treatment patterns
Of the 111 patients in the cohort, 80 (72.1%) are known to have received ART (Table 2). Of the 65 patients who had died, only 10 (15.4%) received dual combination therapy with a NRTI and five (7.7%) combination therapy including a PI or NNRTI, reflecting the lack of treatments available at the time when many of these patients died. The 35 patients who remain alive and who obtain their haemophilia/HIV care at the RFHHC have used a wide range of treatment regimens as ART availability has changed over time. Twenty-eight of these patients (80.0%) have received ART at some stage during infection. There were no significant differences between these 28 patients and the seven who have not received ART in terms of their current age, length of follow-up or previous AIDS diagnosis (Table 3). As expected, those who had received ART had a lower median CD4 cell count at the end of follow-up (P = 0.006) and lower nadir count over follow-up (P = 0.0002) than those who had not received ART. While individuals who had received ART had higher peak RNA levels over follow-up than those who had not received ART (P = 0.008), there were no significant differences between the most recent RNA levels in these two groups (P = 0.83). Of the 28 patients who have received ART, 24 (85.7%) are currently still receiving therapy: 15 are receiving treatment combinations with three or more drugs including a PI or NNRTI, two patients are receiving triple NRTI therapy and seven are receiving dual NRTI therapy.
Response to highly active antiretroviral therapy
Twenty-two patients in the cohort are known to have received HAART including at least one PI or NNRTI while under follow-up at the RFHHC (Table 2). Patients started HAART between November 1995 and March 1999. Eighteen patients received a PI- containing combination (11, ritonavir; 10, saquinavir; 5, nelfinavir; 2, indinavir) and four received an NNRTI-containing regimen (three nevirapine and one loviride). Seventeen patients had taken ART at the time of starting HAART. All of these patients had previously received zidovudine; other antiretroviral drugs previously received were lamivudine (12), didanosine (10), zalcitabine (10) and stavudine (2). Of these 17 patients, 12 added one or more drugs to an existing treatment regimen, four switched all of their existing drugs to drugs that they had never previously received and one switched all drugs but his new combination included a drug to which he had been exposed for a period in the past (zidovudine).
Pretreatment CD4 cell counts were generally low (median 100 × 106 cells/l; range 0-330) and RNA levels high (median 5.13 log10 copies/ml; range 2.60-5.88). One patient had an RNA level of < 400 copies/ml at the time of starting HAART.
Over a median of 464 days (range 44-1248) since starting HAART, 11 patients made changes to their initial HAART regimen by stopping, switching or intensifying their treatment. This first occurred after a median of 116 days (range 7-504). In addition, three patients took a short treatment break before restarting the same HAART regimen. Four patients have died over follow-up; the causes of death in these four patients were cerebral haemorrhage, liver failure/hetatocellular carcinoma, PCP and bronchopneumonia.
Eighteen patients had at least one HIV RNA measurement (median 5 log10 copies/ml; range 1-10) after starting HAART. RNA levels dropped by a median of 0.6 log10 copies/ml in the first 3 months following initiation of HAART and continued to drop thereafter (Fig. 2), although there was wide interindividual variability in the response to HAART. During follow-up, 10 of these 18 patients achieved an RNA level < 400 copies/ml, although in four this occurred after a switch from the initial HAART regimen. After censoring patients at the time of changing their HAART regimen, the Kaplan-Meier rate of having an RNA level < 400 copies/ml was 51.1% by 2 years after starting HAART. At least one CD4 cell count value was obtained in 19 patients after they had started HAART (median 5; range 1-20). CD4 cell counts increased by a median of 95 × 106 cells/l in the period of 3-6 months after starting HAART (Fig. 2), but this initial increase was not maintained and levels fell to approximately 60 × 106 cells/l by 10-12 months after starting HAART.
Clinical side effects of highly active antiretroviral therapy
Unusual bleeds occurred in 8 of the 18 patients receiving a PI. One patient stopped treatment totally; two switched to another PI and one switched to an NNRTI. In all, the bleeding improved following changes in treatment. The remaining four patients remained on the same treatment; in these patients, the bleeding either improved spontaneously or the patients took increased doses of factor VIII prophylaxis. One patient receiving a PI developed diabetes and abnormal lipid levels; an additional patient receiving a PI developed lipomas in both breasts.
Discussion
This cohort is one of the longest groups of prospectively followed HIV seroconverters in the world. Cohorts such as this are extremely useful for documenting the long-term effects of HIV infection, both before and after the introduction of antiretroviral therapy. While a number of patients have transferred their care elsewhere, we have been able to ascertain whether patients were alive for all, and have information on AIDS progression in the majority.
The median time to the development of AIDS in this cohort is now 13.6 years. Similar to observations in other studies of haemophilic individuals [12,13], the AIDS-defining disease tended to be either PCP or oesophageal candidal infection for most patients, although these events occurred fairly early in the epidemic, prior to the introduction of primary prophylaxis against these conditions [14]. No cases of Kaposi's sarcoma have been seen. This condition is rare in haemophilic men [15]; this may be because they are rarely seropositive for human herpesvirus type 8 [16], the possible causal agent for Kaposi's sarcoma.
After an initially high rate in the first few years after infection, the rate of new AIDS-defining diseases remained relatively stable until the end of 1996. Since then there has been a marked decline in the incidence of AIDS in this cohort. The most likely explanation for this is the introduction of HAART from late 1995 onwards. However, we have not seen a similar drop in the death rate, even after taking account of the ageing cohort (data not shown). Many of the deaths in the most recent calendar period were from liver-related causes, almost certainly a consequence of coinfection with HCV. Individuals infected with both HIV and HCV have an increased risk of liver disease [4,5,17]. The reason for this is unclear, although it is thought that infection with HIV may lead to increased HCV loads [18] in these individuals. One alternative explanation for our drop in AIDS rate is that some of the men who would have developed AIDS died from liver-related causes before they could receive an AIDS diagnosis, leading to an underestimate of the AIDS progression rate. Analyses that take account of this possible bias are underway, although as time goes on the increased availability of HAART may mean that AIDS is no longer a suitable endpoint for studies such as these as it fails to capture some of the more relevant clinical effects of HIV infection.
The treatment experience in this cohort reflects the changing treatment patterns over time and as guidelines have changed. Many of the patients who remain alive have progressively used zidovudine or other monotherapy, dual combination therapy and combinations of three or four drugs including either a PI or NNRTI. Some of the patients still follow regimens that we would now consider to be less than optimal: a number of patients remain on double NRTI therapy, for example. The fact that the patients were all infected with HIV and HCV following supposedly safe treatment for haemophilia, and the possibility of spontaneous bleeding following treatment with a PI, has meant that both clinicians and patients are understandably cautious when starting ART.
Few studies have reported response to HAART in patients with haemophilia. Merry et al.[19] reported short-term response to PI in 20 haemophilic patients in Ireland. Our response rates at 3 and 6 months are remarkably similar to those quoted by Merry. RNA changes in the first 3 months after starting HAART were not as large as might be expected, but by 6 months the response rates (possibly after changing treatments) were good. The patients in this cohort were often heavily pretreated with NRTI and were at an advanced disease stage at the time of starting HAART. Therefore, they may be expected to show a less impressive response to HAART than patients in other studies [20,21]. The relative infrequency of RNA monitoring (approximately every 3 months) may also contribute to the slower timing of the response we could detect.
Recently, it has been documented that individuals treated with HAART, and in particular with a PI, may develop lipodystrophy and other metabolic disorders [22,23]. To date, none of our patients have developed clinical features suggestive of lipodystrophy, although one patient has developed diabetes and was found to have abnormal lipid levels. The lack of consensus in the definition of lipodystrophy used in other published studies means that it is difficult to assess whether our rate of abnormalities is any higher or lower than might be expected.
We have previously documented the relationship between PI treatment and unusual bleeding in patients with clotting factor disorders [6]. This bleeding was unpredictable and did not generally respond to increased clotting factor prophylaxis. In the 21 patients who have received HAART in this cohort, eight have now experienced unusual bleeding, all while being treated with a PI. The bleeds resolved in four patients after treatment was changed. The remaining four patients remained on the same treatment and either the bleeds resolved spontaneously or prophylaxis was increased, leading to resolution of the bleeding.
In conclusion, while there has been a reduction in the incidence of AIDS in our cohort, the death rate remains high, a consequence of the large number of deaths related to liver disease now occurring. Many individuals have chosen not to receive HAART or have reduced the number of drugs taken because of worries about toxicities and bleeding. However, where patients are able to maintain their HAART regimen, the long-term virological and immunological response to HAART in this cohort is good.
Acknowledgements
We thank Dr Alec Black, Dr Owen Smith, Dr S. Al-Ismail, Dr Mike Laffan, Dr Martin Fisher, Patricia Lilley and Chris Harrington for providing clinical information about the patients.
In addition, we thank Professor George Janossy and the Department of Immunology for providing CD4 cell count measurements, and Professor Paul Griffiths and the department of Virology for carrying out HIV tests.
References
1. Lee CA, Webster A, Griffiths PD, Kernoff PBA. Symptomless HIV infection after more than ten years [letter]. Lancet 1990, 335: 425 -426.
2. Darby SC, Ewart DW, Giangrande PLF, on behalf of the UK Haemophilia Centre Directors' Organization. Mortality before and after HIV infection in the complete UK population of haemophiliacs. Nature 1995, 377: 79 -82.
3. Kernoff PBA, Lee CA, Karayiannis P, Thomas HC. High risk of non-A non-B hepatitis after a first exposure to volunteer or commercial clotting factor concentrates: effects of prophylactic immune serum globulin. Br J Haematol 1985, 60: 469 -479.
4. Eyster ME, Diamondstone LS, Lien J, Ehmann WC, Quan S, Goedert JJ. Natural history of hepatitis C virus infection in multitransfused hemophiliacs: effect of coinfection with human immunodeficiency virus. J Acquir Immune Defic Syndr 1993, 6: 602 -610.
5. Telfer P, Sabin C, Devereux H, Scott F, Dusheiko G, Lee C. The progression of HCV-associated liver disease in a cohort of haemophilic patients. Br J Haematol 1994, 87: 555 -561.
6. Yee T-T, Armolia P, Lee CA, Mire N, Giangrande PL. Protease inhibitors and unusual bleeding in haemophiliacs [letter]. Haemophilia 1997, 3: 219 -221.
7. Lee CA, Phillips A, Elford J. et al. The natural history of human immunodeficiency virus infection in a haemophilic cohort. Br J Haematol 1989, 73: 228 -234.
8. Sabin CA, Devereux H, Phillips AN, Janossy G, Lee CA, Loveday C. The course of viral load throughout HIV infection. J Acquir Imm Defic Syndr 2000, 23: 172 -177.
9. Ginsburg C, Salmon-Ceron D, Vassilief D. et al. Unusual occurrence of spontaneous haematomas in three asymptomatic HIV-infection haemophila patients a few days after the onset of ritonavir treatment [letter]. AIDS 1997, 11: 388 -389.
10. Ahlbom A. Biostatistics for Epidemiologists. London: Lewis; 1993.
11. SAS Institute:SAS/STAT User's Guide, Version 6. Cary: SAS Institute; 1992.
12. Goedert JJ, Kessler CM, Aledort LM. et al. A prospective study of human immunodeficiency virus type 1 infection and the development of AIDS in subjects with hemophilia. N Engl J Med 1989, 321: 1141 -1148.
13. Astermark J, Johnsson H, Stigendal L, Lethagen S, Berntorp E. HIV disease progression in Swedish haemophiliacs and the influence of replacement therapy. Haemophilia 1997, 3: 277 -282
14. Sabin CA, Elford J, Phillips AN, Janossy G, Lee CA. Prophylaxis forPneumocystis cariniipneumonia: its impact on the natural history of HIV infection in men with haemophilia. Haemophilia 1995, 1: 37 -44.
15. Rabkin CS, Goedert JJ, Biggar RJ, Yellin F, Blattner WA. Kaposi's sarcoma in three HIV-1-infected cohort. J Acquir Imm Defic Syndr 1990, 3 (suppl 1): S38 -S43.
16. Simpson GR, Schultz TF, Whitby D. et al. Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen. Lancet 1996, 348: 1133 -1138.
17. Darby SC, Ewart DW, Giangrande PLF. et al. Mortality from liver cancer and liver disease in haemophilic men and boys in the UK given blood products contaminated with hepatitis C. Lancet 1997, 350: 1425 -1431.
18. Eyster ME, Fried MW, Di Disceglie AM, Goedert JJ. Increasing hepatitis C virus RNA levels in hemophiliacs: relationship to human immunodeficiency virus infection and liver disease. :Multicenter Hemophilia Cohort Study. Blood 1994, 84: 1020 -1023.
19. Merry C, McMahon C, Ryan M, O'Shea E, Mulcahy F, Smith OP. Successful use of protease inhibitors in HIV-infected haemophilia patients. Br J Haematol 1998, 101: 475 -479.
20. Lederman MM, Connick E, Landay A. et al. Immunological responses associated with 12 weeks of combination antiretroviral therapy consisting of zidovudine, lamivudine, and ritonavir: Results of AIDS Clinical Trials Group protocol 315. J Infect Dis 1998, 178: 70 -79.
21. Kaufmann GR, Duncombe C, Cunningham P. et al. Treatment response and durability of a double protease inhibitor therapy with saquinavir and ritonavir in an observational cohort of HIV-1-infected individuals. AIDS 1998, 12: 1625 -1630.
22. Carr A, Samaras K, Thorisdottir A, Kaufmann GR, Chisholm DJ, Cooper DA. Diagnosis, prediction, and natural course of HIV-1 protease-inhibitor-associated lipodystrophy, hyperlipidaemia, and diabetes mellitus: a cohort study. Lancet 1999, 353: 2093 -2099.
23. Walli R, Herfort O, Michl GM. et al. Treatment with protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1-infected patients. AIDS 1998, 12: F167 -F173.
© 2000 Lippincott Williams & Wilkins, Inc.